Targeting the human ccr5 locus as a safe harbor for the expression of therapeutic proteins

ABSTRACT

The present disclosure provides methods and compositions for treating lysosomal storage disorders in subjects, comprising genetically modifying cells from the subjects ex vivo by integrating therapeutic transgenes into the CCR5 locus.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional Pat. Appl. No. 63/044,951, filed on Jun. 26, 2020, which application is incorporated herein by reference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under Grant No. NS102398 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

Lysosomal storage diseases (LSDs) such as Mucopolysaccharidosis type 1. Gaucher disease, and Krabbe disease comprise a large group of genetic disorders caused by deficiencies in lysosomal proteins, and many lack effective treatments. Collectively LSDs have an incidence in the population of about 1 in 7000 births and have severe effects including early death. While clinical trials are in progress on possible treatments for some of these diseases, there is currently no approved treatment for many LSDs. Current treatment options for some but not all LSDs include enzyme replacement therapy (ERT), a medical treatment which replaces an enzyme that is deficient or absent in the body. In some instances, this is done by giving the patient an intravenous (IV) infusion of a solution containing the enzyme. Enzyme replacement therapies, however, can have limited efficacy for various reasons. An alternative approach for treating LSDs would be genome editing. Recently developed genome editing tools combine precise gene addition with genetic alterations that can add therapeutic benefit (16). Among these, Clustered Regularly Interspaced Short Palindromic Repeats-associated protein-9 nuclease (CRISPR/Cas9) is the simplest to engineer and has been used to successfully modify human hematopoietic stem and progenitor cells (HSPCs) in culture (17,18).

This platform consists of two main components: (1) a sgRNA/Cas9 ribonucleoprotein complex (RNP) functioning as an RNA-guided endonuclease, and (2) a designed homologous repair template, delivered using a vector such as adeno-associated viral vector serotype six (AAV6). The RNP can be comprised of a 100-bp, chemically-modified, synthetically-generated, single guide RNA (sgRNA) complexed with Streptococcus pyogenes Cas9-endonuclease and delivered into the cells by, e.g., electroporation (31). In the nucleus, the RNP binds to the target sequence and Cas9 catalyzes a double-stranded break, stimulating one of two repair pathways: 1) non-homologous end joining (NHEJ), in which broken ends are directly ligated, often producing small insertions and deletions (indels); and 2) homology-directed repair (HDR), in which recombination with the supplied homologous repair template is used for precise sequence changes (32). In human hematopoietic stem and progenitor cells (HSPCs), the AAV6 genome is an efficient delivery method for the homologous repair templates containing an experimenter-defined genetic change flanked by homology arms centered at the break site (27). Accordingly, the HDR pathway can be leveraged not only to achieve single-base pair changes, but also to integrate entire expression cassettes, thus enabling stable expression of tailorable combinations of regulatory regions, transgenes, and selectable markers (29,33,34).

Although its therapeutic potential in LSDs remains to be explored, to maximize therapeutic correction by autologous transplantation of genetically modified HSPCs in some LSDs, functional enzymes must sometimes be expressed at higher-than-endogenous levels. This can be achieved by inserting an expression cassette (exogenous promoter-gene of interest) into non-essential genomic region (or “safe harbor”). A safe harbor provides a platform that is independent of specific patient mutations, is easily adaptable to various lysosomal enzymes and, compared to lentiviral transduction, ensures more predictable and consistent transgene expression because the insertion sites are restricted (up to 2 in autosomes). Moreover, its disruption has no effect on cell proliferation and no known potential for oncogenic transformation.

Mucopolysaccharidosis type I (MPSI) is a common LSD caused by insufficient iduronidase (IDUA) activity, which results in glycosaminoglycan (GAG) accumulation and progressive multi-systemic deterioration that severely affects the neurological and musculoskeletal systems (1). Current interventions for MPSI include enzyme replacement therapy (ERT) and allogeneic hematopoietic stem cell transplantation (allo-HSCT); both have limited efficacy. For example, ERT does not cross the blood-brain barrier, requires costly life-long infusions, and inhibitory antibodies can further decrease enzyme bioavailability (2). Allo-HSCT results in better outcomes than ERT by providing a persistent source of enzyme and tissue macrophages that can migrate into affected organs, including the brain, to deliver local enzyme (3,4,5). However, allo-HSCT also has significant limitations, including the uncertain availability of suitable donors, delay in treatment (allowing for irreversible progression), and transplant-associated morbidity and mortality such as graft-versus-host disease and drug-induced immunosuppression.

Human and animal studies in MPSI have shown that the therapeutic efficacy of HSCT can be enhanced by increasing the levels of circulating IDUA. In humans, patients transplanted with non-carrier donors had better clinical responses than patients transplanted with HSPCs from MPSI heterozygotes with decreased enzyme expression (6). In mice, transplantation of virally transduced murine hematopoietic stem and progenitor cells (HSPCs) expressing supra-normal enzyme levels (7,8) dramatically corrected the phenotype. Based on this, autologous transplantation of lentivirus-transduced HSPCs overexpressing lysosomal enzymes is being explored in human trials for LSDs including severe MPSI (ClinicalTrials.gov, NCT03488394) and Metachromatic leukodystrophy (9). The peroxisomal disorder X-linked adrenoleukodystrophy has also been successfully treated by lentiviral transduced autologous HSPCs, though supra-normal expression of the missing enzyme is probably not critical as cross-correction is not a feature of this disease (10,11). This autologous approach eliminates the need to find immunologically matched donors and reduces some of the potential complications from allogeneic transplants. However, concerns remain about the potential for tumorigenicity associated with random insertion of the viral genomes (12,13), carry-over of infectious particles (14), the immune response to some of the vectors, and variable transgene expression (15).

Gaucher Disease (GD) is genetic disorder caused by mutations in the GBA gene that result in glucocerebrosidase (GCase) deficiency and the accumulation of glycolipids in cell types with high glycolipid degradation burden, especially macrophages (1b). GD encompasses a spectrum of clinical findings from a perinatal-lethal form to mildly symptomatic forms. Three major clinical types delineated by the presence (types 2 and 3) or absence (type 1) of central nervous system involvement are commonly used for determining prognosis and management (2b). In western countries, GD type 1 (GD1) is the most common phenotype (˜94% of patients) and typically manifests with hepatosplenomegaly, bone disease, cytopenias, and variably with pulmonary disease, as well as elevated risk for malignancies and Parkinson's disease (3b,4b).

The pathophysiology in GD1 is thought to be driven by glucocerebroside-engorged macrophages that infiltrate the bone marrow, spleen and liver, and promote chronic inflammation as well as low-grade activation of coagulation and complement cascades (5b-7b). Current therapies for GD1 include orally-available small-molecule inhibitors of glucosylceramide synthase (substrate reduction therapy or SRT) and glucocerebrosidase enzyme replacement (ERT) targeted to macrophages via mannose receptor-mediated uptake (8b-13b). While ameliorative for visceral and skeletal disease manifestations, these therapies are chronically administered, life-long, and costly. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been applied successfully as a one-time treatment for GD1 (14b) and its therapeutic effect is likely achieved through supplying graft-derived GCase-competent macrophages. However, because of the significant transplant-related morbidity and mortality of allo-HSCT. ERT and SRT are standard of care for patients with GD1 (15b,16b).

The effectiveness of macrophage-targeted ERT and allo-HSCT for treating GD1 suggests that restoration of GCase function in macrophages alone is sufficient for phenotypic correction in GD1. Consequently, restoring GCase activity in the patient's own hematopoietic system to establish an autologous approach that averts many of the risks of allo-HSCT could be a safer and potentially curative therapy for this disease. Furthermore, unlike ERT and the best tolerated SRT, it could provide enzyme reconstitution in the brain that could benefit neuronopathic forms of the disease (14b). For these reasons, non-targeted gene addition into human hematopoietic stem and progenitor cells (HSPCs) have been explored, first using retroviruses (17b-20b) and later lentiviral vectors, and have yielded promising results in murine GD models (21b-23b). Nevertheless, concerns remain about the potential for insertional mutagenesis and malignant transformation in viral gene transfer (24b,25b) stressing the need for the development of “targeted” gene addition strategies to generate genetically modified HSPCs for human therapy.

Krabbe disease (also called globoid cell leukodystrophy) is an inherited neurological disorder, one of a group of disorders called leukodystrophies, that results from the loss of myelin (demyelination) of nerve cells in the nervous system. Krabbe disease can begin in infants (infantile form) or in childhood, adolescence, or adulthood (late-onset forms) and is caused by a deficiency in galactocerebrosidase (GALC) enzyme activity.

There is a need for new, safe, and effective approaches for introducing transgenes into cells encoding therapeutic proteins for the treatment of lysosomal storage disorders, for example into autologous HSPCs in vivo or ex vivo. The present disclosure satisfies this need and provides other advantages as well.

BRIEF SUMMARY

The present disclosure provides methods and compositions for treating lysosomal storage disorders (LSDs), in particular through the genetic modification of cells taken from a subject with an LSD in order to introduce a functional copy of a therapeutic gene into the cells, and subsequently reintroducing the modified cells back into the subject. In particular, the present methods and compositions involve the homologous-recombination mediated introduction of therapeutic transgenes into the genome of cells at the CCR5 locus.

Accordingly, in one aspect, the present disclosure provides a method of genetically modifying a cell from a subject with a lysosomal storage disorder (LSD), the method comprising: introducing into a cell isolated from the subject a single guide RNA (sgRNA) targeting the CCR5 locus, an RNA-guided nuclease, and a homologous donor template comprising a transgene encoding a therapeutic protein that is absent or deficient in the subject, wherein: the sgRNA binds to the nuclease and directs it to a target sequence at the CCR5 locus in the genome comprising the sequence shown as SEQ ID NO:3 or SEQ ID NO:4, whereupon the nuclease cleaves the CCR5 locus at the target sequence, wherein: the homologous donor template comprises a first homology region comprising the sequence of SEQ ID NO:1 or a fragment thereof to one side of the transgene, and a second homology region comprising the sequence of SEQ ID NO:2 or a fragment thereof to the other side of the transgene, and the transgene is integrated into the genome by homology directed recombination (HDR) at the site of the cleaved CCR5 locus, and wherein: the integrated transgene directs the expression of the therapeutic protein in the cell.

In some embodiments, the method further comprises isolating the cell from the subject prior to the introducing of the sgRNA, RNA-guided nuclease, and homologous donor template. In some embodiments, the sgRNA comprises chemical modifications at one or more nucleotides. In some such modifications, the sgRNA comprises 2′-O-methyl-3′-phosphorothioate (MS) modifications at one or more nucleotides. In some such embodiments, the 2′-O-methyl-3′-phosphorothioate (MS) modifications are present at the three terminal nucleotides of the 5′ and 3′ ends. In some embodiments, the target sequence of sgRNA comprises the sequence of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the sgRNA comprises the sequence of SEQ ID NO:5. In some embodiments, the RNA-guided nuclease is Cas9. In some embodiments, the sgRNA and the RNA-guided nuclease are introduced into the cell as a ribonucleoprotein (RNP). In some embodiments, the RNP is introduced into the cell by electroporation. In some embodiments, the transgene is present within an expression cassette. In some such embodiments, the expression cassette comprises a coding sequence for the therapeutic protein, operably linked to a promoter, and an exogenous polyadenylation signal. In some embodiments, the polyadenylation signal is a bovine growth hormone polyadenylation signal. In some embodiments, the homologous donor template is introduced into the cells using a recombinant adeno-associated virus (rAAV) vector. In some such embodiments, the recombinant adeno-associated virus is serotype 6 (rAAV6).

In some embodiments, the LSD is mucopolysaccharidosis type 1, and the therapeutic protein is iduronidase. In some embodiments, the transgene is part of an expression cassette comprising the coding sequence for iduronidase, operably linked to a phosphoglycerate kinase (PGK) promoter or a spleen focus-forming virus (SFFV) promoter. In some embodiments, the homologous donor template comprises the sequence of SEQ ID NO: 6 or SEQ ID NO: 7. In some embodiments, the cell is a CD34⁺ hematopoietic stem and progenitor cell (HSPC). In some embodiments, the LSD is Gaucher disease, and the therapeutic protein is glucocerebrosidase. In some such embodiments, the transgene is part of an expression cassette comprising the coding sequence for glucocerebrosidase, operably linked to a CD68 promoter or derivative thereof. In some embodiments, the donor template comprises the sequence of SEQ ID NO: 8. In some embodiments, the cell is a CD34⁺ hematopoietic stem and progenitor cell (HSPC). In some embodiments, the LSD is Krabbe disease, and the therapeutic protein is galactocerebrosidase. In some embodiments, the transgene is part of an expression cassette comprising the coding sequence for galactocerebrosidase, operably linked to a CD68 promoter or a derivative thereof. In some such embodiments, the cell is a CD34⁺ hematopoietic stem and progenitor cell (HSPC) or a neuronal stem cell.

In another aspect, the present disclosure provides a method of treating a subject in need thereof with a lysosomal storage disorder, comprising (i) genetically modifying a cell from the subject using any of the herein-described methods, and (ii) reintroducing the cell into the subject, wherein the reintroducing is effective to treat the subject.

In some embodiments, the cell is reintroduced into the subject by systemic transplantation. In some embodiments, the cell is reintroduced into the subject by local transplantation. In some embodiments, the transplantation is intrafemoral or intrahepatic. In some embodiments, the cell is cultured, selected, and/or induced to undergo differentiation in vitro prior to being reintroduced into the subject.

In another aspect, the present disclosure provides an sgRNA that specifically targets the CCR5 gene, wherein the target sequence of the sgRNA comprises the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:4.

In some embodiments, the sgRNA comprises the nucleotide sequence of SEQ ID NO:5. In some embodiments, the sgRNA comprises chemical modifications at one or more nucleotides. In some such embodiments, the sgRNA comprises 2′-O-methyl-3′-phosphorothioate (MS) modifications at one or more nucleotides. In some embodiments, the 2′-O-methyl-3′-phosphorothioate (MS) modifications are present at the three terminal nucleotides of the 5′ and 3′ ends.

In another aspect, the present disclosure provides a homologous donor template comprising: (i) an expression cassette comprising: (a) a coding sequence for a therapeutic protein, operably linked to (b) a promoter, and (c) a polyadenylation signal at the 3′ end of the coding sequence; (ii) a first CCR5 homology region located to one side of the expression cassette within the donor template, wherein the first CCR5 homology region comprises SEQ ID NO:1 or a fragment thereof; and (iii) a second CCR5 homology region located to the other side of the expression cassette within the donor template, wherein the second CCR5 homology region comprises SEQ ID NO:2 or a fragment thereof.

In some embodiments, the therapeutic protein is iduronidase. In some embodiments, the therapeutic protein is glucocerebrosidase. In some embodiments, the therapeutic protein is galactocerebrosidase. In some embodiments, the donor template comprises the sequence of SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.

In another aspect, the present invention provides an HSPC comprising any of the herein-described sgRNAs and/or homologous donor templates.

In another aspect, the present disclosure provides a genetically modified HSPC comprising an integrated transgene at the CCR5 locus, wherein the integrated transgene comprises a coding sequence for iduronidase, glucocerebrosidase, or galactocerebrosidase. In some embodiments, the HSPC was modified using any of the herein-described methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-ID. Efficient CRISPR/Cas9-mediated integration of IDUA overexpression cassettes into the CCR5 locus in human CD34⁺ HSPCs. FIG. 1A: Schematic of targeted integration of IDUA and expression cassettes. The AAV6 genome was constructed to have 500 bp arms of homology centered on the cut site, and the IDUA sequence placed under the control of the SFFV or the PGK promoter (E=Exon). In two DNA templates, YFP was expressed downstream of IDUA using the self-cleaving P2A peptide. FIG. 1B: Representative FACs and histogram plots 3-days post-modification of mock and human HSPCs that underwent RNP and AAV6 exposure with YFP-containing expression cassettes. FIG. 1C: Targeting frequencies in cord blood (CB, red dots) and adult peripheral blood (PB, blue dots)-derived HSPCs read by percent fluorescent cells in YFP-expressing cassettes and percent colonies with targeted CCR5 alleles by single cell-derived colony genotyping in cassettes without the reporter. Each dot represents the average of duplicates for a human cell donor. For RNP+AAV6 conditions with YFP templates, n=20 and n=11 independent human donors for CB and PB respectively. For the template without selection n=6 independent human donors in CB and PB. Data shown as mean±SD. FIG. 1D: Distribution of wild type (WT), mono and bi-allelically modified cells (n=400) in YFP-positive HSPCs

FIGS. 2A-2F. Enhanced IDUA expression by IDUA-HSPCs and derived macrophages. FIG. 2A: Representative FACS plot showing three distinct populations based on YFP expression 3 days post-modification. FIG. 2B: Percent YFP-positive cells in culture (30 days). FIG. 2C: Fold increase in IDUA secretion and intracellular expression by YFP-high, YFP-low, and YFP-negative populations compared to mock cells. FIG. 2D: Average LAMP-1+area in MPSI fibroblasts co-cultured with IDUA-HSPCs. Each dot represents a cell. FIG. 2E: Human CD34, CD14, and CD11b marker expression in HSPC-derived macrophages after in vitro differentiation compared to undifferentiated cells (CD34⁺ HSPCs). Macrophage morphology and YFP expression after differentiation. FIG. 2F: Fold increase in IDUA secretion and intracellular expression in HSPC-macrophages modified with SFFV and PGK expression cassettes. FIGS. 2C, 2E, 2F: Each column represents average of triplicates in n=3 independent biological samples. All data expressed as mean±SD, ***p<001 in two-sided unpaired t-test. Source data are provided as a Source Data file.

FIGS. 3A-3G. IDUA-HSPCs maintain long-term repopulation capacity. FIG. 3A: Schematic and representative FACS plots showing phenotyping by flow of human, myeloid, B-cell, and targeted cells after engraftment. FIG. 3B: Percent human cell chimerism in bone marrow (BM) and peripheral blood (PM) of mice 16-weeks post-transplant with CB (blue dots) and PB (red dots)-derived HSPCs targeted with PGK-IDUA-YFP cassette. Each point represents an individual mouse; mock (n=11), YFP− (n=21), and YFP+ (n=36). FIG. 3C: Percent human, YFP+ cells in BM of mice in BM 16-weeks post-transplant. FIG. 3D: Percent human cell chimerism in BM in mice transplanted with bulk cells without selection with two different human cell donors; donor 1 n=9, donor 2 n=5. FIG. 3E: Percent modified alleles in engrafted cells by ddPCR. 28% was the starting allele modification frequency for both human donors. FIG. 3F: Percent human cell chimerism in BM of mice in secondary transplants 32 weeks after genome editing; YFP− (n=10), and YFP+ (n=10). FIG. 3G: Percent human, YFP+ cells in BM of mice in secondary transplants.

FIGS. 4A-4F. Biochemical correction in NSG-IDUAX/X mice by human IDUA-HSPCs. IDUA activity and GAG accumulation in heterozygous sham-treated (W/X sham-clear), heterozygous transplanted (W/X Tx-black), homozygous sham-treated (X/X sham-blue), and homozygous transplanted (X/X Tx-red) mice. FIG. 4A: Percent human and YFP+ cells in BM in experiments using bulk and sorted cells. FIG. 4B: Urinary GAGs at 4, 8, and 18 weeks in experiments using bulk cells (n=5 mice per cohort, two measurements per mouse). FIG. 4C: Plasma and tissue IDUA activity in experiments using bulk cells (n=5 per cohort). FIG. 4D: Fold GAG storage in liver, spleen, and brain (normalized by W/X sham, n=5 per cohort). FIG. 4E: Plasma and tissue IDUA activity in experiments using sorted cells (n=5 for W/X Tx and sham mice, and n=13 for X/X Tx and sham mice). FIG. 4F: Fold GAG urinary excretion and tissue storage in experiments using sorted cells (normalized by W/X sham). Median values shown in all scatter plots. FIGS. 4D, 4F show box plots with whiskers at the 5-95th percentiles. ****p<0.0001 in one-way ANOVA test. Post hoc comparisons were made with the Tukey's multiple comparisons test.

FIGS. 5A-5I. Phenotypic reconstitution in NSG-IDUAX/X mice by human IDUA-HSPCs. FIG. 5A: Representative photos showing facial features in mice transplanted with bulk cells. FIG. 5B: Bony features in mice transplanted with bulk cells (W/X sham (clear), X/X sham (blue), and X/X Tx (red), n=5 mice per cohort. Box plots with whiskers show median, min and max. FIG. 5C: Bony features in mice transplanted with sorted cells (W/X sham (clear or gray, n=11), X/X sham (blue, n=10), and X/X Tx (red, n=11). FIG. 5D: Ambulatory distance in mice transplanted with sorted cells. W/X sham vs. X/X sham: **; W/X sham vs. X/X Tx: n.s.; X/X sham vs. X/X Tx: *. FIG. 5E: Vertical rearing in mice transplanted with sorted cells. W/X sham vs. X/X sham: *; W/X sham vs. X/X Tx: n.s.; X/X sham vs. X/X Tx: *. FIG. 5F: Memory retention in mice transplanted with sorted cells. FIG. 5G: Quantification of digging behavior in mice transplanted with sorted cells. FIGS. 5H-5I: Measurement of neuroinflammation in the cerebral cortex, n=3 mice in five sections. FIG. 5H: Microglia (Isolectin B4, n=15 brain sections from three independent mice, and (FIG. 5I) astrocytes (GFAP, n=15 brain sections from 3 independent mice. For FIGS. 5D-5G, data shown as mean±SEM. For FIGS. 5H-5I: data shown as mean±SD. All comparisons between groups were performed using one-way ANOVA test and post hoc comparisons were made with the Tukey's multiple comparisons test. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Open field testing and vertical rearings were analyzed using within-subject modeling by calculating the area under the curve for each mouse within the first five minutes and comparing between groups with one-way ANOVA. Source data are provided as a Source Data file

FIG. 6 . OFF-target analysis of the CCR5 sgRNA. Percent reads with Indels at 62 off-target sites (OT) predicted using COSMID. For each site, red dots indicate samples treated with WT Cas9 and blue dots indicate samples treated with HiFi Cas9. The limit of detection for NGS is 0.1% and is indicated on the graph by a dashed line.

FIGS. 7A-7E. Characterization of the CCR5 sgRNA. FIG. 7A: Indel frequency in Cord blood (CB, red) and adult peripheral blood (PB, blue)-derived cells by the RNP complex. Data expressed as mean±SD. CB n=13 and PB n=13. Each dot represents a different biological sample. FIG. 7B: Representative indel distribution from next generation sequencing reads. FIG. 7C: Representative histogram of CCR5 protein expression in mock and RNP-treated cells showing an 80% reduction in protein expression after RNP electroporation. FIG. 7D: Sample sequence traces around the CCR5 sgRNA sequence (gray box, PAM in red) in mock samples and RNP-treated CB-derived HSPCs showing predominant single A insertion. FIG. 7E: Representative summary of indels with frequencies greater than 0.1%.

FIGS. 8A-8E. Efficiency of modification at the CCR5 locus. FIG. 8A: Schematic showing the three primer-based genotyping scheme to distinguish mono and bi-allelic integration into the CCR5 locus on CFA-derived colonies. This strategy did not distinguish WT versus alleles with indels (NHEJ). FIG. 8B: Example agarose gels of 40 colonies genotyped in this manner. A single 1.1 Kb band was interpreted as WT/NHEJ in both alleles, while a single 0.6 Kb band was read as bi-allelic integration. FIG. 8C: Schematic of probe design for ddPCR analysis. Fraction of modified alleles was obtained by using a second reference probe to the CCRL2 gene also on chromosome 3p. FIG. 8D: Two probes where each straddled a 5′ or 3′ homology arm were designed. The accuracy of the assays was compared using genomic DNA from colonies derived from mono-allelic cells (0.5 fraction of alleles modified). Error bars indicate 95% CI. The 3′ HA probe was selected. FIG. 8E: CCR5 allele targeting frequencies in CB (red n=20) and PB (blue n=11)-derived IDUA expressing HSPCs as measured by ddPCR. For the template without selection CB=6, PB=6. Data shown as mean±SD. Source data are provided as a Source Data file.

FIGS. 9A-9E. Efficient targeting of GCase to the (CCR5 locus in human HSPCs 48-hours post-modification. FIG. 9A: Schematic of gene targeting mediated by sgRNA/Cas9 RNP and rAAV targeting vectors. FIG. 9B: Schematic of expected CD68S promoter activation. FIG. 9C: Representative flow plots of Citrine expression versus forward scatter (FSC) for HSPCs without treatment (mock), treated with rAAV alone (AAV), and treated with RNP and rAAV (RNP+AAV). FIG. 9D: Flow cytometric quantification of Citrine+HSPCs targeted with SFFV-GCase-P2A-Citrine and CD68S-GCase-P2A-Citrine vectors (n=9). FIG. 9E: Percent of CCR5 alleles with integrated CD68S-GBA-P2A-Citrine and SFFV-GBA-P2A-Citrine cassettes in AAV only (white), bulk (black), FACS-enriched Citrine− (gray) and Citrine+ (green) HSPCs, and in CD68S-GCase-targeted unselected cells (black). Data shown as mean±SD.

FIGS. 10A-10F. Generation of human GCase-macrophages from genome edited HSPCs FIG. 10A: Representative images showing phase contrast, phagosomes visualized by pHrodo-labeled E. coli, and nuclei in mock-treated human HSPCs after 20 days in macrophage differentiation media. FIG. 10B: Human CD34, CD14, and CD11b marker expression in HSPC-derived macrophages and human monocyte-derived macrophages after in vitro differentiation compared to undifferentiated cells (CD34⁺ HSPCs). FIG. 10C: Representative images showing phase contrast, Citrine expression, phagosomes visualized by pHrodo-labeled E. coli, and nuclei in mock-treated, SFFV-GCase-P2A-Citrine, and CD68S-GCase-P2A-Citrine targeted macrophages. FIG. 10D: Human CD14, and CD11b marker expression in the same cells with and without in vitro differentiation. Left graph: CD11b+. Middle graph: CD14⁺. Right graph: CD11b+/CD14⁺. FIG. 10E: Representative FACS plots of FMO's and Mock sample showing CD11b and CD14 expression in HSPC maintenance or Macrophage differentiation media. FIG. 10F: Representative FACS plots showing CD11b and CD14 expression in CD68S-GCase-Citrine+ and SFFV-GCase-Citrine+ cells in HSPC maintenance or macrophage differentiation media. Data shown as mean±SD (n=3).

FIGS. 11A-11F. The CD68S promoter confines GCase expression to the monocyte/macrophage lineage. FIG. 11A: Representative flow plots depicting Citrine+ and Citrine− populations at the time of sort (day 0, 48-h post-modification) and after 20 days in HSPC maintenance (HSPC) or macrophage differentiation (MΦ) cultures. FIG. 11B: Citrine expression expressed as % Citrine+ cells over time in HSPC and MΦ cultures (n=3 biological replicates). FIG. 11C: Citrine expression expressed MFI over time in HSPC and MG cultures in the CD68S-GCase-P2A-Citrine-targeted cells. FIG. 11D: Fold GCase activity in HSPC and FIG. 11E: MΦ cultures in targeted cells compared to unmodified (mock-treated) cells (n=3). Comparisons between groups were performed using one-way ANOVA test and post-hoc comparisons were made with the Tukey's multiple comparisons test. **: p<0.01. FIG. 11F: Percent of targeted CCR5 alleles at the time of sort and after 20 days in HSPC and MΦ cultures (n=3). Data shown as mean±SD. Source data are provided as a Source Data file.

FIGS. 12A-12G. GCase-targeted HSPCs sustain long-term hematopoiesis. FIG. 12A: Total number of colonies formed from mock, Citrine+ and Citrine− SFFV and CD68S-driven constructs. FIG. 12B: Distribution of phenotypes of colonies formed. Erythroid progenitors (burst forming unit-erythroid or BFU-E (red)) and colony-forming unit-erythroid or CFU-E (blue), granulocyte-macrophage progenitors (CFU-GM, green), and multi-potential granulocyte, erythroid, macrophage, megakaryocyte progenitor cells (CFU-GEMM, purple). FIG. 12C: Primary human engraftment (16-wks) in the bone marrow in transplants using CD68S-GCase-targeted and CD68S-GCase-P2A-Citrine-targeted cells (Blue circles: 0.25E6, green: 1E6, and red: 2E6 cells transplanted; n=31,33). FIG. 12D: Primary human engraftment in the spleen. FIG. 12E: Targeted allele frequency in CD68S-GCase- and CD68S-GCase-P2A-Citrine-targeted cells before transplantation (Pre-Tx) and 16-wks post-transplantation (Post-Tx) in engrafted human cells in bone marrow of mice with human chimerism >1% (n=29,31). FIG. 12F: Secondary human engraftment (32-wks) in the bone marrow (n=8, 3 with chimerism <1%). FIG. 12G: Targeted allele frequency before (Pre-Tx) and after transplant (Post-Tx) in the bone marrow cells of secondary mice. FIGS. 12A-12B: Data shown as mean±SD. (c-g) Medians shown.

FIGS. 13A-13E. In vivo monocyte/macrophage lineage differentiation of GCase-targeted HSPCs. FIG. 13A: Distribution of B-lymphoid and myeloid lineage cells within the engrafted human cell population in the bone marrow from mice transplanted with CD68S-GCase and CD68S-GCase-P2A-Citrine-targeted HSPCs (n=29, 31). FIG. 13B: Distribution of B-lymphoid and lineage cells within the engrafted human cell population from secondary transplants. Empty: vector without Citrine (n=5). FIG. 13C: Representative FACS plots showing Citrine expression in human CD33+ (myeloid), CD14⁺ (monocyte) and CD19 (B-cells). FIG. 13D: Percent Citrine positive cells in monocyte, myeloid, and B-cell populations in mice with human CCR5 allele modification fraction>10%. FIG. 13E: Representative epifluorescence microscopy images of in vitro generation of human CD68S-GBA1-P2A-Citrine-targeted macrophages from sorted CD14⁺ monocytes. Images depict morphology (brightfield), nuclei (Hoechst), CD68S (red), and Citrine (green). FIGS. 13A, 13B, 13D: Median shown. Source data are provided as a Source Data file.

FIGS. 14A-14H. Improved macrophage differentiation of GCase-targeted HSPCs in NSG-SGM3 mice. FIG. 14A: Human cell engraftment 16-weeks post-transplantation in the bone marrow (BM), spleen (SP), and peripheral blood (PB) in transplants using CD68S-GCase-P2A-Citrine-targeted cells (n=5). FIG. 14B: Modified allele frequency from engrafted CD68S-GCase-P2A-Citrine-targeted cells (n=5). FIG. 14C: Percent human B-cell (CD19⁺), myeloid (CD33⁺), and monocyte (CD14⁺) populations in BM, SP, and PB shown in white. Citrine positive cells in each population are shown in green. FIG. 14D: Representative FACS plots showing CD45⁺, CD45⁺/CD11b⁺ and CD45⁺/CD11b⁺/Citrine populations in macrophage preparations from lung, peritoneal macrophages, and liver. FIG. 14E: Percent human CD45⁺ and human CD45⁺/Citrine cells (n=5). FIG. 14F: Percent human CD45⁺/CD11b⁺ and human CD45⁺/CD11b⁺/Citrine cells (n=5). FIG. 14G: Fold GCase activity in human Citrine+ cells compared to human Citrine− cells in BM, SP, and lung from three different mice FIG. 14H: Modified allele frequency in human Citrine⁺ cells compared to human Citrine⁻ cells in BM, SP, and lung from three different mice.

DETAILED DESCRIPTION 1. Introduction

The present disclosure provides methods and compositions for the treatment of lysosomal storage disorders in subjects through the introduction and integration at the CCR5 locus of transgenes encoding therapeutic proteins. The methods involve the introduction of ribonucleoproteins (RNPs) comprising single guide RNAs (sgRNAs) and RNA-guided nucleases (e.g., Cas9) into cells from the subject, as well as the introduction of homologous templates for repair. In particular, the methods and compositions can be used to efficiently introduce and express functional transgenes encoding enzymes that are deficient in the subject. In particular embodiments, the RNP complexes, e.g., comprising CCR5 sgRNA and Cas9 protein, are delivered to cells via electroporation, followed by the transduction of the homologous template using an AAV6 viral vector. The homologous templates for repair are constructed to have arms of homology centered on the cut site, located on either side of the coding sequence for a therapeutic protein of interest, under the control of a designated promoter. Transcription is terminated using an exogenous polyadenylation signal. Depending on the promoter, the system can achieve, e.g., supraphysiological expression and/or cell-specific expression. This system can be used to modify any human cell.

2. General

Practicing this invention utilizes routine techniques in the field of molecular biology. Basic texts disclosing the general methods of use in this invention include Sambrook and Russell, Molecular Cloning. A Laboratory Manual (3rd ed. 2001): Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994)).

For nucleic acids, sizes are given in either kilobases (kb), base pairs (bp), or nucleotides (nt). Sizes of single-stranded DNA and/or RNA can be given in nucleotides. These are estimates derived from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences. For proteins, sizes are given in kilodaltons (kDa) or amino acid residue numbers. Protein sizes are estimated from gel electrophoresis, from sequenced proteins, from derived amino acid sequences, or from published protein sequences.

Oligonucleotides that are not commercially available can be chemically synthesized, e.g., according to the solid phase phosphoramidite triester method first described by Beaucage and Caruthers, Tetrahedron Lett. 22:1859-1862 (1981), using an automated synthesizer, as described in Van Devanter et. al., Nucleic Acids Res. 12:6159-6168 (1984). Purification of oligonucleotides is performed using any art-recognized strategy, e.g., native acrylamide gel electrophoresis or anion-exchange high performance liquid chromatography (HPLC) as described in Pearson and Reanier, J Chrom. 255: 137-149 (1983).

3. Definitions

As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

The terms “a,” “an,” or “the” as used herein not only include aspects with one member, but also include aspects with more than one member. For instance, the singular forms “a,” “an.” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells, and so forth.

The terms “about” and “approximately” as used herein shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typically, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Any reference to “about X” specifically indicates at least the values X, 0.8X, 0.81X, 0.82X, 0.83X, 0.84X, 0.85X, 0.86X, 0.87X, 0.88X, 0.89X, 0.9X, 0.91X, 0.92X, 0.93X, 0.94X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, 1.05X, 1.06X, 1.07X, 1.08X, 1.09X, 1.1X, 1.11X, 1.12X, 1.13X, 1.14X, 1.15X, 1.16X, 1.17X, 1.18X, 1.19X, and 1.2X. Thus, “about X” is intended to teach and provide written description support for a claim limitation of, e.g., “0.98X.”

The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

The term “gene” means the segment of DNA involved in producing a polypeptide chain. It may include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).

A “promoter” is defined as an array of nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. The promoter can be a heterologous promoter.

An “expression cassette” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular polynucleotide sequence in a host cell. An expression cassette may be part of a plasmid, viral genome, or nucleic acid fragment. Typically, an expression cassette includes a polynucleotide to be transcribed, operably linked to a promoter. The promoter can be a heterologous promoter. In the context of promoters operably linked to a polynucleotide, a “heterologous promoter” refers to a promoter that would not be so operably linked to the same polynucleotide as found in a product of nature (e.g., in a wild-type organism).

As used herein, a first polynucleotide or polypeptide is “heterologous” to an organism or a second polynucleotide or polypeptide sequence if the first polynucleotide or polypeptide originates from a foreign species compared to the organism or second polynucleotide or polypeptide, or, if from the same species, is modified from its original form. For example, when a promoter is said to be operably linked to a heterologous coding sequence, it means that the coding sequence is derived from one species whereas the promoter sequence is derived from another, different species, or, if both are derived from the same species, the coding sequence is not naturally associated with the promoter (e.g., is a genetically engineered coding sequence).

“Polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. All three terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

The terms “expression” and “expressed” refer to the production of a transcriptional and/or translational product, e.g., of a therapeutic protein and/or a nucleic acid sequence encoding a therapeutic protein. In some embodiments, the term refers to the production of a transcriptional and/or translational product encoded by a gene (e.g., a iduronidase, glucocerebrosidase, or galactocerebrosidase gene) or a portion thereof. The level of expression of a DNA molecule in a cell may be assessed on the basis of either the amount of corresponding mRNA that is present within the cell or the amount of protein encoded by that DNA produced by the cell.

A “therapeutic protein” as used herein refers to a protein or a functional fragment thereof, encoded by a “therapeutic gene”, that is deficient in a patient with a lysosomal storage disorder (LSD) or whose expression would be beneficial in a patient with an LSD. Typically, the therapeutic protein is a lysosomal enzyme, but any secreted protein that would be beneficial for a patient with an LSD can be used. In particular embodiments, the therapeutic protein is iduronidase (in particular for patients with mucopolysaccharidosis type I, which is caused by mutations in the IDUA gene), glucocerebrosidase (in particular for patients with Gaucher disease, which is caused by mutations in the GBA gene), or galactocerebrosidase (in particular for patients with Krabbe disease, which is caused by mutations in the GALC gene).

The term “treating” or “treatment” refers to any one of the following: ameliorating one or more symptoms of a disease or condition (e.g., a lysosomal storage disorder); preventing the manifestation of such symptoms before they occur; slowing down or completely preventing the progression of the disease or condition (as may be evident by longer periods between reoccurrence episodes, slowing down or prevention of the deterioration of symptoms, etc.), enhancing the onset of a remission period; slowing down the irreversible damage caused in the progressive-chronic stage of the disease or condition (both in the primary and secondary stages); delaying the onset of said progressive stage: or any combination thereof.

As used herein, the terms “subject”, “individual” or “patient” refer, interchangeably, to a warm-blooded animal such as a mammal. In particular embodiments, the term refers to a human. A subject may have, be suspected of having, or be predisposed to a lysosomal storage disorder as described herein. The term also includes livestock, pet animals, or animals kept for study, including horses, cows, sheep, poultry, pigs, cats, dogs, zoo animals, goats, primates (e.g. chimpanzee), and rodents. A “subject in need thereof” refers to a subject that has one or more symptoms of a lysosomal storage disorder (LSD), that has received a diagnosis of an LSD, that is suspected of having or being predisposed to a LSD, that shows a deficiency of one or more therapeutic proteins as described herein, or that is thought to potentially benefit from increased expression of a therapeutic protein as described herein.

An “effective amount” refers to an amount of a compound or composition, as disclosed herein effective to achieve a particular biological, therapeutic, or prophylatic result. Such results include, without limitation, the treatment of a disease or condition disclosed herein as determined by any means suitable in the art.

“Iduronidase” is an enzyme (see, e.g., UniProt ID P35475 for human Alpha-L-iduronidase), encoded by the IDUA gene (see, e.g., NCBI Gene ID 3425 for human IDUA), that hydrolyzes the terminal alpha-L-iduronic acid residues of two glycosaminoglycans, dermatan sulfate and heparan sulfate. This hydrolysis reaction is required for the degradation of these glycosaminoglycans in lysosomes. Mutations in this gene that result in enzymatic deficiency lead to, e.g., mucopolysaccharidosis type I (MPS I), a lysosomal storage disorder (LSD) as described herein. Any iduronidase enzyme, from any source, or any polynucleotide encoding an iduronidase enzyme, can be used in the present methods, so long that it is capable of hydrolyzing terminal alpha-L-iduronic acid residues and restoring or increasing enzyme function in cells, e.g., cells of a subject with MPS I. In particular embodiments, the iduronidase used in the present methods is encoded by a polynucleotide comprising nucleotides 1002-2960 of SEQ ID NO:6 or SEQ ID NO:7, or to a functional iduronidase encoded by a polynucleotide comprising a sequence with, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to nucleotides 1002-2960 of SEQ ID NO:6 or SEQ ID NO:7.

“Glucocerebrosidase” or “beta-glucocerebrosidase” or “glucosylceramidase beta” is a lysosomal enzyme (see, e.g., UniProt ID P04062 for human glucocerebrosidase/glucosylceramidase), encoded by the GBA gene (see, e.g., NCBI Gene ID 2629 for human GBA), that hydrolyzes glucosylceramide into free ceramide and glucose. Mutations in this gene that result in enzymatic deficiency lead, e.g., to Gaucher disease, a lysosomal storage disorder (LSD) as described herein that involves an accumulation of glucocerebrosides. Any glucocerebrosidase or glucosylceramidase enzyme, from any source, or any polynucleotide encoding a glucocerebrosidase or glucosylceramidase enzyme, can be used in the present methods, so long that it is capable of hydrolyzing a beta-glucosidic linkage in glucosylceramide and thereby restoring or increasing enzyme function in cells. e.g., cells of a subject with Gaucher disease. In particular embodiments, the iduronidase used in the present methods is encoded by a polynucleotide comprising nucleotides 982-2589 of SEQ ID NO:8, or to a functional iduronidase encoded by a polynucleotide comprising a sequence with, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to nucleotides 982-2589 of SEQ ID NO:8.

“Galactocerebrosidase” or “galactoceramidase” is a lysosomal enzyme (see, e.g., UniProt ID P54803 for human galactocerebrosidase/galactoceramidase), encoded by the GALC gene (see, e.g., NCBI Gene ID 2581 for human GALC), that hydrolyzes galactoester bonds of glycolipids such as galactosylceramide and galctosylsphingosine. Mutations in this gene that result in enzymatic deficiency lead, e.g., to Krabbe disease, a lysosomal storage disorder (LSD) as described herein. Any galactocerebrosidase or galactoceramidase enzyme, from any source, or any polynucleotide encoding a galactocerebrosidase or galactoceramidase enzyme, can be used in the present methods, so long that it is capable of hydrolyzing galactoester bonds of glycolipids and thereby restoring or increasing enzyme function in cells, e.g., cells of a subject with Krabbe disease.

“Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, “conservatively modified variants” refers to those nucleic acids that encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles. In some cases, conservatively modified variants of a therapeutic protein can have an increased stability, assembly, or activity as described herein.

The following eight groups each contain amino acids that are conservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K): 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W): 7) Serine (S), Threonine (T): and 8) Cysteine (C), Methionine (M)

(see, e.g., Creighton. Proteins. W. H. Freeman and Co., N. Y. (1984)).

Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

In the present application, amino acid residues are numbered according to their relative positions from the left most residue, which is numbered 1, in an unmodified wild-type polypeptide sequence.

As used in herein, the terms “identical” or percent “identity,” in the context of describing two or more polynucleotide or amino acid sequences, refer to two or more sequences or specified subsequences that are the same. Two sequences that are “substantially identical” have at least 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithm or by manual alignment and visual inspection where a specific region is not designated. With regard to polynucleotide sequences, this definition also refers to the complement of a test sequence. With regard to amino acid sequences, in some cases, the identity exists over a region that is at least about 50 amino acids or nucleotides in length, or more preferably over a region that is 75-100 amino acids or nucleotides in length.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. For sequence comparison of nucleic acids and proteins, the BLAST 2.0 algorithm and the default parameters discussed below are used.

A “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

An algorithm for determining percent sequence identity and sequence similarity is the BLAST 2.0 algorithm, which is described in Altschul et al., (1990) J. Mol. Biol. 215: 403-410. Software for performing BLAST analyses is publicly available at the National Center for Biotechnology Information website, ncbi.nlm.nih.gov. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits acts as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T. and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=−2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sd. USA 89:10915 (1989)).

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

The “CRISPR-Cas” system refers to a class of bacterial systems for defense against foreign nucleic acids. CRISPR-Cas systems are found in a wide range of bacterial and archaeal organisms. CRISPR-Cas systems fall into two classes with six types, I, II, III, IV, V, and VI as well as many sub-types, with Class 1 including types I and III CRISPR systems, and Class 2 including types II, IV, V and VI: Class 1 subtypes include subtypes I-A to I-F, for example. See, e.g., Fonfara et al., Nature 532, 7600 (2016); Zetsche et al., Cell 163, 759-771 (2015); Adli et al. (2018). Endogenous CRISPR-Cas systems include a CRISPR locus containing repeat clusters separated by non-repeating spacer sequences that correspond to sequences from viruses and other mobile genetic elements, and Cas proteins that carry out multiple functions including spacer acquisition, RNA processing from the CRISPR locus, target identification, and cleavage. In class I systems these activities are effected by multiple Cas proteins, with Cas3 providing the endonuclease activity, whereas in class 2 systems they are all carried out by a single Cas, Cas9.

A “homologous repair template” refers to a polynucleotide sequence that can be used to repair a double stranded break (DSB) in the DNA, e.g., a CRISPR/Cas9-mediated break at the CCR5 locus as induced using the herein-described methods and compositions. The homologous repair template comprises homology to the genomic sequence surrounding the DSB, i.e., comprising CCR5 homology arms of the invention. In some embodiments, two distinct homologous regions are present on the template, with each region comprising at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 or more nucleotides or more of homology with the corresponding genomic sequence. In particular embodiments, the templates comprise two homology arms comprising about 500 nucleotides of homology extending from either site of the sgRNA target site. The repair template can be present in any form, e.g., on a plasmid that is introduced into the cell, as a free floating doubled stranded DNA template (e.g., a template that is liberated from a plasmid in the cell), or as single stranded DNA. In particular embodiments of the present invention, the template is present within a viral vector. e.g., an adeno-associated viral vector such as AAV6. In particular embodiments, the templates comprise an expression cassette comprising a sequence encoding a therapeutic protein, e.g., iduronidase, glucocerebrosidase, or galactocerebrosidase, operably linked to a promoter, such that the expression cassette is integrated into the genome at the CCR5 locus and the therapeutic protein is expressed.

As used herein, “homologous recombination” or “HR” refers to insertion of a nucleotide sequence during repair of double-strand breaks in DNA via homology-directed repair mechanisms. This process uses a “donor template” or “homologous repair template” with homology to nucleotide sequence in the region of the break as a template for repairing a double-strand break. The presence of a double-stranded break facilitates integration of the donor sequence. The donor sequence may be physically integrated or used as a template for repair of the break via homologous recombination, resulting in the introduction of all or part of the nucleotide sequence. This process is used by a number of different gene editing platforms that create the double-strand break, such as meganucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR-Cas9 gene editing systems. In particular embodiments of the present invention, HR involves double-stranded breaks induced by CRISPR-Cas9.

A “lysosomal storage disorder” or “LSD” refers to an inherited metabolic disease characterized by an abnormal build-up of various toxic materials in the body's cells as a result of enzyme deficiencies. There are nearly 50 of these disorders altogether, and they affect different parts of the body, including the skeleton, brain, skin, heart, and central nervous system. Non-limiting examples include Sphingolipidoses, Farber disease (ASAH1 deficiency), Krabbe disease (galactosylceramidase or GALC deficiency), Galactosialidosis, Gangliosidoses, Alpha-galactosidase, Fabry disease (α-galactosidase deficiency-GLA, or agalsidase alpha/beta), Schindler disease (alpha-NAGA deficiency), GM1 gangliosidosis, GM2 gangliosidoses (beta-hexosaminidase deficiency), Sandhoff disease (hexosaminidase-B deficiency), Tay-Sachs disease (hexosaminidase-A deficiency). Gaucher's disease Type 1/2/3 (glucocerebrosidase deficiency-gene name: GBA), Wolman disease (LAL deficiency), Niemann-Pick disease type A/B (sphingomyelin phosphodiesterase ldeficiency—SMPD1 or acid sphingomyelinase). Sulfatidosis, Metachromatic leukodystrophy, Hurler syndrome (alpha-L iduronidase deficiency—IDUA), Hunter syndrome or MPS2 (iduronate-2-sulfatase deficiency-idursulfase or IDS), Sanfilippo syndrome, Morquio, Maroteaux-Lamy syndrome, Sly syndrome (β-glucuronidase deficiency). Mucolipidosis, I-cell disease. Lipidosis. Neuronal ceroid lipofuscinoses, Batten disease (tripeptidyl peptidase-I deficiency), Pompe (alglucosidase alpha deficiency), hypophosphatasia (asfotase alpha deficiency), MPSI (laronidase deficiency), MPS3A (heparin N-sulfatase deficiency), MPS3B (alpha-N-acetylglucosaminidase deficiency), MPS3C (heparin-a-glucosaminide N-acetyltransferase deficiency), MPS3D (N-acetylglucosamine 6-sulfatase deficiency), MPS4 (elosulfase alpha deficiency), MPS6 (glasulfate deficiency), MPS7 (B-glucoronidase deficiency), phenylketonuria (phenylalanine hydroxylase deficiency), and MLD (arylsulphatase A deficiency). In particular embodiments, the LSD treated using the present methods and compositions is Mucopolysaccharidosis type 1, Gaucher Disease, or Krabbe Disease.

4. CRISPR/Cas Systems Targeting the CCR5 Safe Harbor Locus

The present disclosure provides methods and compositions for integrating and expressing transgenes encoding therapeutic proteins, e.g., therapeutic proteins such as iduronidase, glucocerebrosidase, or galactocerebrosidase, into the CCR5 safe harbor locus in cells from a subject with a lysosomal storage disorder (LSD). In particular embodiments, the cells are hematopoietic stem and progenitor cells (HSPCs) or neuronal stem cells. The cells can be modified using the methods described herein and then reintroduced into the subject, wherein the expression of the therapeutic protein in the modified cells in vivo can restore enzyme activity that is missing or deficient in the subject with the LSD.

The present invention is based in part on the identification of CRISPR guide sequences that specifically direct the cleavage of CCR5, e.g., within exon 3 of CCR5, by RNA-guided nucleases such as Cas9. In particular embodiments, the methods involve the introduction of ribonucleoproteins (RNPs) comprising an sgRNA targeting CCR5 and Cas9, as well as a template DNA molecule comprising CCR5 homology arms flanking the transgene encoding the therapeutic protein. Using the present methods, high rates of targeted integration at the CCR5 locus and expression of the therapeutic gene can be achieved, with the result that the transplantation and long-term engraftment of the modified cells can lead to a reduction or elimination of symptoms caused by the enzyme deficiency associated with the LSD.

sgRNAs

The single guide RNAs (sgRNAs) used in the present invention target the CCR5 locus. sgRNAs interact with a site-directed nuclease such as Cas9 and specifically bind to or hybridize to a target nucleic acid within the genome of a cell, such that the sgRNA and the site-directed nuclease co-localize to the target nucleic acid in the genome of the cell. The sgRNAs as used herein comprise a targeting sequence comprising homology (or complementarity) to a target DNA sequence at the CCR5 locus, and a constant region that mediates binding to Cas9 or another RNA-guided nuclease. The sgRNA can target any sequence within CCR5 adjacent to a PAM sequence. In some embodiments, the target sequence is within exon 3 of CCR5. In particular embodiments, the target sequence of the sgRNA comprises the sequence shown as SEQ ID NO: 3 or SEQ ID NO: 4. In particular embodiments, the sgRNA comprises the sequence shown as SEQ ID NO:5, or a sequence having, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO:5, or comprising, e.g., 1, 2, 3 or more nucleotide substitutions in SEQ ID NO:5.

The targeting sequence of the sgRNAs may be, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length, or 15-25, 18-22, or 19-21 nucleotides in length, and shares homology with a targeted genomic sequence, in particular at a position adjacent to a CRISPR PAM sequence. The sgDNA targeting sequence is designed to be homologous to the target DNA, i.e., to share the same sequence with the non-bound strand of the DNA template or to be complementary to the strand of the template DNA that is bound by the sgRNA. The homology or complementarity of the targeting sequence can be perfect (i.e., sharing 100% homology or 100% complementarity to the target DNA sequence) or the targeting sequence can be substantially homologous (i.e., having less than 100% homology or complementarity, e.g., with 1-4 mismatches with the target DNA sequence).

Each sgRNA also includes a constant region that interacts with or binds to the site-directed nuclease, e.g., Cas9. In the nucleic acid constructs provided herein, the constant region of an sgRNA can be from about 70 to 250 nucleotides in length, or about 75-100 nucleotides in length, 75-85 nucleotides in length, or about 80-90 nucleotides in length, or 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more nucleotides in length. The overall length of the sgRNA can be, e.g., from about 80-3(0) nucleotides in length, or about 80-150 nucleotides in length, or about 80-120 nucleotides in length, or about 90-110 nucleotides in length, or, e.g. 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 nucleotides in length.

It will be appreciated that it is also possible to use two-piece gRNAs (cr:tracrRNAs) in the present methods, i.e., with separate crRNA and tracrRNA molecules in which the target sequence is defined by the crispr RNA (crRNA), and the tracrRNA provides a binding scaffold for the Cas nuclease.

In some embodiments, the sgRNAs comprise one or more modified nucleotides. For example, the polynucleotide sequences of the sgRNAs may also comprise RNA analogs, derivatives, or combinations thereof. For example, the probes can be modified at the base moiety, at the sugar moiety, or at the phosphate backbone (e.g., phosphorothioates). In some embodiments, the sgRNAs comprise 3′ phosphorothiate internucleotide linkages, 2′-O-methyl-3′-phosphoacetate modifications, 2′-fluoro-pyrimidines, S-constrained ethyl sugar modifications, or others, at one or more nucleotides. In particular embodiments, the sgRNAs comprise 2′-O-methyl-3′-phosphorothioate (MS) modifications at one or more nucleotides (see, e.g., Hendel et al. (2015) Nat. Biotech. 33(9):985-989, the entire disclosure of which is herein incorporated by reference). In particular embodiments, the 2′-O-methyl-3′-phosphorothioate (MS) modifications are at the three terminal nucleotides of the 5′ and 3′ ends of the sgRNA.

The sgRNAs can be obtained in any of a number of ways. For sgRNAs, primers can be synthesized in the laboratory using an oligo synthesizer, e.g., as sold by Applied Biosystems, Biolytic Lab Performance, Sierra Biosystems, or others. Alternatively, primers and probes with any desired sequence and/or modification can be readily ordered from any of a large number of suppliers, e.g., ThermoFisher, Biolytic, IDT, Sigma-Aldritch, GeneScript, etc.

RNA-Guided Nuclease

Any CRISPR-Cas nuclease can be used in the method, i.e., a CRISPR-Cas nuclease capable of interacting with a guide RNA and cleaving the DNA at the target site as defined by the guide RNA. In some embodiments, the nuclease is Cas9 or Cpf1. In particular embodiments, the nuclease is Cas9. The Cas9 or other nuclease used in the present methods can be from any source, so long that it is capable of binding to an sgRNA of the invention and being guided to and cleaving the specific CCR5 sequence targeted by the targeting sequence of the sgRNA. In particular embodiments, Cas9 is from Streptococcus pyogenes.

Also disclosed herein are CRISPR/Cas or CRISPR/Cpf1 systems that target and cleave DNA at the CCR5 locus. An exemplary CRISPR/Cas system comprises (a) a Cas (e.g., Cas9) or Cpf1 polypeptide or a nucleic acid encoding said polypeptide, and (b) an sgRNA that hybridizes specifically to CCR5, or a nucleic acid encoding said guide RNA. In some instances, the nuclease systems described herein, further comprises a donor template as described herein. In particular embodiments, the CRISPR/Cas system comprises an RNP comprising an sgRNA targeting CCR5 and a Cas protein such as Cas9. In some embodiments, the Cas9 is a high fidelity (HiFi) Cas9 (37).

In addition to the CRISPR/Cas9 platform (which is a type II CRISPR/Cas system), alternative systems exist including type I CRISPR/Cas systems, type III CRISPR/Cas systems, and type V CRISPR/Cas systems. Various CRISPR/Cas9 systems have been disclosed, including Streptococcus pyogenes Cas9 (SpCas9), Streptococcus thermophilus Cas9 (StCas9), Campylobacter jejuni Cas9 (CjCas9) and Neisseria cinerea Cas9 (NcCas9) to name a few. Alternatives to the Cas system include the Francisella novicida Cpf1 (FnCpf1), Acidaminococcus sp. Cpf1 (AsCpf1), and Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1) systems. Any of the above CRISPR systems may be used to induce a single or double stranded break at the CCR5 locus to carry out the methods disclosed herein.

Introducing the sgRNA and Cas Protein into Cells

The sgRNA and nuclease can be introduced into a cell using any suitable method, e.g., by introducing one or more polynucleotides encoding the sgRNA and the nuclease into the cell, e.g., using a vector such as a viral vector or delivered as naked DNA or RNA, such that the sgRNA and nuclease are expressed in the cell. In particular embodiments, the sgRNA and nuclease are assembled into ribonucleoproteins (RNPs) prior to delivery to the cells, and the RNPs are introduced into the cell by, e.g., electroporation. RNPs are complexes of RNA and RNA-binding proteins. In the context of the present methods, the RNPs comprise the RNA-binding nuclease (e.g., Cas9) assembled with the guide RNA (e.g., sgRNA), such that the RNPs are capable of binding to the target DNA (through the gRNA component of the RNP) and cleaving it (via the protein nuclease component of the RNP). As used herein, an RNP for use in the present methods can comprise any of the herein-described guide RNAs and any of the herein-described RNA-guided nucleases.

Animal cells, mammalian cells, preferably human cells, modified ex vivo, in vitro, or in vivo are contemplated. Also included are cells of other primates; mammals, including commercially relevant mammals, such as cattle, pigs, horses, sheep, cats, dogs, mice, rats; birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and/or turkeys.

In some embodiments, the cell is an embryonic stem cell, a stem cell, a progenitor cell, a pluripotent stem cell, an induced pluripotent stem (iPS) cell, a somatic stem cell, a differentiated cell, a mesenchymal stem cell or a mesenchymal stromal cell, a neural stem cell, a hematopoietic stem cell or a hematopoietic progenitor cell, an adipose stem cell, a keratinocyte, a skeletal stem cell, a muscle stem cell, a fibroblast, an NK cell, a B-cell, a T cell, or a peripheral blood mononuclear cell (PBMC). In particular embodiments, the cells are hematopoietic stem and progenitor cells (HSPCs), e.g., cord blood-derived (CB) or adult peripheral blood-derived (PB) HSPCs, or neuronal stem cells.

To avoid immune rejection of the modified cells when administered to a subject, the cells to be modified are preferably derived from the subject's own cells. Thus, preferably the mammalian cells are autologous cells from the subject to be treated with the modified cells. In some embodiments, however, the cells are allogeneic, i.e., isolated from an HLA-matched or HLA-compatible, or otherwise suitable, donor.

In some embodiments, cells are harvested from the subject and modified according to the methods disclosed herein, which can include selecting certain cell types, optionally expanding the cells and optionally culturing the cells, and which can additionally include selecting cells that contain the transgene integrated into the CCR5 locus. In some embodiments, the cells are induced to undergo differentiation, e.g., into macrophages or monocytes, using methods known in the art and as described herein. In some embodiments, such modified, selected, and/or differentiated cells are then reintroduced into the subject.

Further disclosed herein are methods of using said nuclease systems to produce the modified host cells described herein, comprising introducing into the cell (a) an RNP of the invention that targets and cleaves DNA at the CCR5 locus, and (b) a homologous donor template or vector as described herein. Each component can be introduced into the cell directly or can be expressed in the cell by introducing a nucleic acid encoding the components of said one or more nuclease systems.

Such methods will target integration of the transgene encoding the therapeutic protein to the CCR5 locus in a host cell ex vivo. Such methods can further comprise (a) introducing a donor template or vector into the cell, optionally after expanding said cells, or optionally before expanding said cells, and (b) optionally culturing the cell.

In some embodiments, the disclosure herein contemplates a method of producing a modified mammalian host cell, the method comprising introducing into a mammalian cell: (a) an RNP comprising a Cas nuclease such as Cas9 and an sgRNA specific to the CCR5 locus, and (b) a homologous donor template or vector as described herein.

In any of these methods, the nuclease can produce one or more single stranded breaks within the CCR5 locus, or a double stranded break within the CCR5 locus. In these methods, the CCR5 locus is modified by homologous recombination with said donor template or vector to result in insertion of the transgene into the locus. The methods can further comprise (c) selecting cells that contain the transgene integrated into the CCR5 locus

Techniques for insertion of transgenes, including large transgenes, capable of expressing functional proteins, including enzymes, cytokines, antibodies, and cell surface receptors are known in the art. (See, e.g. Bak and Porteus, Cell Rep. 2017 Jul. 18; 20(3): 750-756 (integration of EGFR); Kanojia et al., Stem Cells. 2015 October; 33(10):2985-94 (expression of anti-Her2 antibody); Eyquem et al., Nature. 2017 Mar. 2; 543(7643):113-117 (site-specific integration of a CAR); O'Connell et al., 2010 PLoS ONE 5(8): e12009 (expression of human IL-7); Tuszynski et al., Nat Med. 2005 May; 11(5):551-5 (expression ofNGF in fibroblasts); Sessa et al., Lancet. 2016 Jul. 30:388(10043):476-87 (expression of arylsulfatase A in ex vivo gene therapy to treat MLD); Rocca et al., Science Translational Medicine 25 Oct. 2017: Vol. 9, Issue 413, eaaj2347 (expression of frataxin); Bak and Porteus, Cell Reports, Vol. 20, Issue 3, 18 Jul. 2017, Pages 750-756 (integrating large transgene cassettes into a single locus), Dever et al., Nature 17 Nov. 2016: 539, 384-389 (adding tNGFR into hematopoietic stem cells (HSC) and HSPCs to select and enrich for modified cells); each of which is hereby incorporated by reference in its entirety.)

Homologous Repair Templates

The transgene to be integrated is typically present within a homologous repair template, or homologous donor template. The transgene can be any transgene whose gene product has a beneficial effect in subjects with a lysosomal storage disorder. In particular embodiments, the transgene is used to replace or compensate for a defective or deficient gene, e.g., a defective iduronidase (IDUA) gene in a subject with Mucopolysaccharidosis type 1, a defective glucocerebrosidase (GBA) gene in a subject with Gaucher disease, or a defective galactocerebrosidase (GALC) gene in a subject with Krabbe disease.

In particular embodiments, the transgene is flanked in the template by CCR5 homology regions. For example, an exemplary template can comprise, in linear order: a first CCR5 homology region, a promoter, a coding sequence for a therapeutic protein, a polyA sequence such as a bovine growth hormone polyadenylation sequence (bGH-PolyA), and a second CCR5 homology region, where the first and second homology regions are homologous to the genomic sequences extending in either direction from the sgRNA target site. In particular embodiments, one of the homology regions comprises the sequence of SEQ ID NO:1, and the other homology region comprises the sequence of SEQ ID NO:2, and/or to a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater identity to SEQ ID NO:1 and/or SEQ ID NO:2.

In one embodiment, for the treatment of mucopolysaccharidosis type 1, the therapeutic protein is iduronidase, and the promoter is the phosphoglycerate kinase (PGK) promoter or the spleen focus-forming virus (SFFV) promoter. This system can be used to modify any human cell. In particular embodiments, the system is used to genetically modify human CD34⁺ hematopoietic stem and progenitor cells. In some embodiments, the homologous repair template comprises the sequence shown as SEQ ID NO: 6 or SEQ ID NO:7, or a derivative or fragment of SEQ ID NO:6 or SEQ ID NO:7, e.g., a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater identity to SEQ ID NO:6 or SEQ ID NO:7 or to a fragment thereof.

In another embodiment, for the treatment of Gaucher disease, the protein is glucocerebrosidase, and the promoter is the CD68 promoter, e.g., the human CD68 promoter. In some embodiments, the promoter is a shortened derivative of the human CD68 promoter, with expression restricted to the monocyte/macrophage lineage. This system can be used to modify any human cell. In particular embodiments, the system is used to genetically modify human CD34⁺ hematopoietic stem and progenitor cells. In some embodiments, the homologous repair template comprises the sequence shown as SEQ ID NO: 8, or a derivative or fragment of SEQ ID NO:8, e.g., a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater identity to SEQ ID NO:8 or to a fragment thereof.

In another embodiment, for the treatment of Krabbe disease, the protein is galactocerebrosidase, and the promoter is CD68, e.g., a shortened derivative of the human CD68 promoter with expression restricted to the monocyte/macrophage lineage. As such, expression of the enzyme can be induced primarily in monocytes/macrophages. This system can be used to modify any human cell. In particular embodiments, the system is used to genetically modify human neuronal stem cells or human CD34⁺ hematopoietic stem and progenitor cells.

In addition to the promoters disclosed above, any promoter that can induce expression of the therapeutic protein in the modified cells can be used, including endogenous and heterologous promoters, inducible promoters, constitutive promoters, cell-specific promoters, and others. In some instances, in addition to the promoter, the transgene is optionally linked to one or more regulatory elements such as enhancers or post-transcriptional regulatory sequences. For example, one can include regulatory sequences (microRNA (miRNA) target sites) in the RNA to avoid expression in certain tissues (post-transcriptional targeting). In some instances, the expression control sequence functions to express the therapeutic transgene following the same expression pattern as in normal individuals (physiological expression) (See Toscano et al., Gene Therapy (2011) 18, 117-127 (2011), incorporated herein by reference in its entirety for its references to promoters and regulatory sequences).

Constitutive mammalian promoters include, but are not limited to, the promoters for the following genes: hypoxanthine phosphoribosyl transferase (HPTR), adenosine deaminase, pyruvate kinase, α-actin promoter and other constitutive promoters. Exemplary viral promoters which function constitutively in eukaryotic cells include, for example, promoters from the simian virus, papilloma virus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, the long terminal repeats (LTR) of Moloney leukemia virus and other retroviruses, and the thymidine kinase promoter of herpes simplex virus. Commonly used promoters including the CMV (cytomegalovirus) promoter/enhancer, EF1a (elongation factor 1a), SV40 (simian virus 40), chicken β-actin and CAG (CMV, chicken β-actin, rabbit β-globin), Ubiquitin C and PGK, all of which provide constitutively active, high-level gene expression in most cell types. Other constitutive promoters are known to those of ordinary skill in the art.

Inducible promoters are activated in the presence of an inducing agent. For example, the metallothionein promoter is activated to increase transcription and translation in the presence of certain metal ions. Other inducible promoters include alcohol-regulated, tetracycline-regulated, steroid-regulated, metal-regulated, nutrient-regulated promoters, and temperature-regulated promoters.

Tissue-specific and/or physiologically regulated expression can also be pursued by modifying mRNA stability and/or translation efficiency (post-transcriptional targeting) of the transgenes. Alternatively, the incorporation of miRNA target recognition sites (miRTs) into the expressed mRNA has been used to recruit the endogenous host cell machinery to block transgene expression (detargeting) in specific tissues or cell types. miRNAs are noncoding RNAs, approximately 22 nucleotides, that are fully or partially complementary to the 3′ UTR region of particular mRNA, referred to as miRTs. Binding of a miRNA to its particular miRTs promotes translational attenuation/inactivation and/or degradation. Regulation of expression through miRNAs is described in Geisler and Fechner, World J Exp Med. 2016 May 20, 6(2): 37-54; Brown and Naldini, Nat Rev Genet. 2009 August, 10(8):578-85; Gentner and Naldini, Tissue Antigens. 2012 November, 80(5):393-403.

To facilitate homologous recombination, the transgene is flanked within the polynucleotide or donor construct by sequences homologous to the target genomic sequence, i.e., CCR5. In particular, the transgene is flanked by sequences surrounding the site of cleavage as defined by sgRNA. In a particular embodiment, the transgene is flanked on one side by a sequence comprising SEQ ID NO:1 or a fragment thereof, and on the other side by a sequence comprising SEQ ID NO:2 or a fragment thereof. The homology regions can be of any size, e.g., 50-2000, 100-1500 bp, 300-900 bp, 400-600 bp, or about 50, 100, 200, 300, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500 or more bp.

Any suitable method can be used to introduce the polynucleotide, or donor construct, into the cell. In particular embodiments, the polynucleotide is introduced using a recombinant adeno-associated viral vector (rAAV). For example, the rAAV can be from serotype 1 (e.g., an rAAV1 vector), 2 (e.g., an rAAV2 vector), 3 (e.g., an rAAV3 vector), 4 (e.g., an rAAV4 vector), 5 (e.g., an rAAV5 vector), 6 (e.g., an rAAV6 vector), 7 (e.g., an rAAV7 vector), 8 (e.g., an rAAV8 vector), 9 (e.g., an rAAV9 vector), 10 (e.g., an rAAV10 vector), or 11 (e.g., an rAAV11 vector). In particular embodiments, the vector is an rAAV6 vector. In some instances, the donor template is single stranded, double stranded, a plasmid or a DNA fragment. In some instances, plasmids comprise elements necessary for replication, including a promoter and optionally a 3′ UTR.

Further disclosed herein are vectors comprising (a) one or more nucleotide sequences homologous to the CCR5 locus, and (b) a transgene encoding a therapeutic factor of the invention. The vector can be a viral vector, such as a retroviral, lentiviral (both integration competent and integration defective lentiviral vectors), adenoviral, adeno-associated viral or herpes simplex viral vector. Viral vectors may further comprise genes necessary for replication of the viral vector.

In some embodiments, the targeting construct comprises: (1) a viral vector backbone, e.g. an AAV backbone, to generate virus; (2) arms of homology to the target site of at least 200 bp but ideally at least 400 bp on each side to assure high levels of reproducible targeting to the site (see, Porteus, Annual Review of Pharmacology and Toxicology, Vol. 56:163-190 (2016); which is hereby incorporated by reference in its entirety); (3) a transgene encoding a therapeutic protein and capable of expressing the therapeutic protein; (4) an expression control sequence operably linked to the transgene; and optionally (5) an additional marker gene to allow for enrichment and/or monitoring of the modified host cells. Any AAV known in the art can be used. In some embodiments the primary AAV serotype is AAV6.

Suitable marker genes are known in the art and include Myc, HA, FLAG, GFP, truncated NGFR, truncated EGFR, truncated CD20, truncated CD19, as well as antibiotic resistance genes (e.g., pac (puromycin-N-acetyl transferase), aph (aminoglycoside phosphotransferase), or hsd (blasticidin S deaminase), providing resistance to puromycin, G418, and blasticidin, respectively).

In any of the preceding embodiments, the donor template or vector comprises a nucleotide sequence homologous to a fragment of the CCR5 locus, optionally to the sequences shown as SEQ ID NO:1 and/or SEQ ID NO:2 or fragments thereof, wherein the nucleotide sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 98%, or 99% identical to at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1000 or more consecutive nucleotides of the CCR5 locus, e.g., to SEQ ID NO:1 and/or SEQ ID NO:2.

The inserted construct can also include other safety switches, such as a standard suicide gene into the locus (e.g. iCasp9) in circumstances where rapid removal of cells might be required due to acute toxicity. The present disclosure provides a robust safety switch so that any engineered cell transplanted into a body can be eliminated, e.g., by removal of an auxotrophic factor. This is especially important if the engineered cell has transformed into a cancerous cell.

5. Methods of Treatment

Following the integration of the transgene into the genome of the cell, e.g., HSPC, and confirming expression of the encoded therapeutic protein, a plurality of modified cells can be reintroduced into the subject, such that they can repopulate and differentiate into, e.g., macrophages or monocytes, and due to the expression of the integrated transgene, can improve one or more abnormalities or symptoms in the subject with the LSD. In some embodiments, the cells are expanded, selected, or induced to undergo differentiation, prior to reintroduction into the subject.

Disclosed herein, in some embodiments, are methods of treating an LSD in an individual in need thereof, the method comprising providing to the individual enzyme replacement therapy using the genome modification methods disclosed herein. In some instances, the method comprises a modified host cell ex vivo, comprising a transgene encoding an enzyme, i.e., therapeutic protein, integrated at the CCR5 locus, wherein said modified host cell expresses an enzyme that is deficient in the individual, thereby treating the LSD in the individual. In some instances, the enzyme is iduronidase, e.g., when the subject has mucopolysaccharidosis type 1. In some instances, the enzyme is glucocerebrosidase, e.g., when the subject has Gaucher disease. In some instances, the enzyme is galactocerebrosidase, e.g. when the subject has Krabbe disease.

In some embodiments, the genetically modified cells express the therapeutic protein (e.g. iduronidase, glucocerebrosidase, or galactocerebrosidase) at a level that is at least, e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more or more of a level representative of a healthy individual without an LSD. In some embodiments, tissues of the subject, e.g., in plasma, liver, spleen, brain, comprise an enzymatic activity provided by the genetically modified transplanted cells, that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, or more of a level representative of a healthy individual without an LSD.

In some embodiments, the guide RNA displays off-target activity (e.g., >0.1% indels) at less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 location. In particular embodiments, the off-target activity occurs at less than 4, 3, 2, or 1 location. In particular embodiments, the off-target activity occurs at 1 or 0 locations when a HiFi Cas9 is used.

In some embodiments, following introduction of the guide RNA, RNA-guided nuclease, and donor template, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more of the targeted cells comprise an integrated transgene. In some embodiments, following transplantation of the genetically modified cells, chimerism in the subject is at least about 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (e.g., 100%).

Pharmaceutical Compositions

Disclosed herein, in some embodiments, are methods, compositions and kits for use of the modified cells, including pharmaceutical compositions, therapeutic methods, and methods of administration. Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any animals.

In some embodiments, a pharmaceutical composition comprising a modified autologous host cell of the invention is provided. The modified autologous host cell is genetically engineered to comprise an integrated transgene encoding the therapeutic protein at the CCR5 locus. The modified host cell of the disclosure herein may be formulated using one or more excipients to, e.g.: (1) increase stability; (2) alter the biodistribution (e.g., target the cell line to specific tissues or cell types); (3) alter the release profile of an encoded therapeutic factor.

Formulations of the present disclosure can include, without limitation, saline, liposomes, lipid nanoparticles, polymers, peptides, proteins, and combinations thereof. Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. As used herein the term “pharmaceutical composition” refers to compositions including at least one active ingredient (e.g., a modified host cell) and optionally one or more pharmaceutically acceptable excipients. Pharmaceutical compositions of the present disclosure may be sterile.

Relative amounts of the active ingredient (e.g. the modified host cell), a pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure may vary, depending upon the identity, size, and/or condition of the subject being treated and further depending upon the route by which the composition is to be administered. For example, the composition may include between 0.1% and 99% (w/w) of the active ingredient. By way of example, the composition may include between 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5-80%, or at least 80% (w/w) active ingredient.

Excipients, as used herein, include, but are not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and the like, as suited to the particular dosage form desired. Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporated herein by reference in its entirety). The use of a conventional excipient medium may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium may be incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition.

Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and/or combinations thereof.

Injectable formulations may be sterilized, for example, by filtration through a bacterial-retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

Dosing and Administration

The modified host cells of the present disclosure included in the pharmaceutical compositions described above may be administered by any delivery route, systemic delivery or local delivery, which results in a therapeutically effective outcome. These include, but are not limited to, enteral, gastroenteral, epidural, oral, transdermal, intracerebral, intracerebroventricular, epicutaneous, intradermal, subcutaneous, nasal, intravenous, intra-arterial, intramuscular, intracardiac, intraosseous, intrathecal, intraparenchymal, intraperitoneal, intravesical, intravitreal, intracavernous), interstitial, intra-abdominal, intralymphatic, intramedullary, intrapulmonary, intraspinal, intrasynovial, intrathecal, intratubular, parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, soft tissue, and topical. In particular embodiments, the cells are transplanted intrafemorally or intrahepatically. In certain embodiments, the composition may take the form of solid, semi-solid, lyophilized powder, or liquid dosage forms, such as, for example, tablets, pills, pellets, capsules, powders, solutions, suspensions, emulsions, suppositories, retention enemas, creams, ointments, lotions, gels, aerosols, foams, or the like, preferably in unit dosage forms suitable for simple administration of precise dosages.

In some embodiments, a subject will undergo a conditioning regime before cell transplantation. For example, before hematopoietic stem cell transplantation, a subject may undergo myeloablative therapy, non-myeloablative therapy or reduced intensity conditioning to prevent rejection of the stem cell transplant even if the stem cell originated from the same subject. The conditioning regime may involve administration of cytotoxic agents. The conditioning regime may also include immunosuppression, antibodies, and irradiation. Other possible conditioning regimens include antibody-mediated conditioning (see, e.g., Czechowicz et al., 318(5854) Science 1296-9 (2007); Palchaudari et al., 34(7) Nature Biotechnology 738-745 (2016); Chhabra et al., 10:8(351) Science Translational Medicine 351ra105 (2016)) and CAR T-mediated conditioning (see, e.g., Arai et al., 26(5) Molecular Therapy 1181-1197 (2018); each of which is hereby incorporated by reference in its entirety). For example, conditioning needs to be used to create space in the brain for microglia derived from engineered hematopoietic stem cells (HSCs) to migrate in to deliver the protein of interest (as in recent gene therapy trials for ALD and MLD). The conditioning regimen is also designed to create niche “space” to allow the transplanted cells to have a place in the body to engraft and proliferate. In HSC transplantation, for example, the conditioning regimen creates niche space in the bone marrow for the transplanted HSCs to engraft. Without a conditioning regimen, the transplanted HSCs cannot engraft.

Certain aspects of the present disclosure are directed to methods of providing pharmaceutical compositions including the modified host cell of the present disclosure to target tissues of mammalian subjects, by contacting target tissues with pharmaceutical compositions including the modified host cell under conditions such that they are substantially retained in such target tissues. In some embodiments, pharmaceutical compositions including the modified host cell include one or more cell penetration agents, although “naked” formulations (such as without cell penetration agents or other agents) are also contemplated, with or without pharmaceutically acceptable excipients.

The present disclosure additionally provides methods of administering modified host cells in accordance with the disclosure to a subject in need thereof. The pharmaceutical compositions including the modified host cell, and compositions of the present disclosure may be administered to a subject using any amount and any route of administration effective for preventing, treating, or managing the LSD. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. The subject may be a human, a mammal, or an animal. The specific therapeutically or prophylactically effective dose level for any particular individual will depend upon a variety of factors including the disorder being treated and the severity of the disorder: the activity of the specific payload employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration: the duration of the treatment; drugs used in combination or coincidental with the specific modified host cell employed; and like factors well known in the medical arts.

In certain embodiments, modified host cell pharmaceutical compositions in accordance with the present disclosure may be administered at dosage levels sufficient to deliver from, e.g., about 1×10⁴ to 1×10⁵, 1×10⁵ to 1×10⁶, 1×10⁶ to 1×10⁷, or more modified cells to the subject, or any amount sufficient to obtain the desired therapeutic or prophylactic, effect. The desired dosage of the modified host cells of the present disclosure may be administered one time or multiple times. In some embodiments, delivery of the modified host cell to a subject provides a therapeutic effect for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or more than 10 years.

The modified host cells may be used in combination with one or more other therapeutic, prophylactic, research or diagnostic agents, or medical procedures, either sequentially or concurrently. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent.

Use of a modified mammalian host cell according to the present disclosure for treatment of a lysosomal disease, disorder or condition is also encompassed by the disclosure.

The present disclosure also contemplates kits comprising compositions or components of the invention, e.g., sgRNA, Cas9, RNPs, and/or homologous templates, as well as, optionally, reagents for, e.g., the introduction of the components into cells. The kits can also comprise one or more containers or vials, as well as instructions for using the compositions in order to modify cells and treat subjects according to the methods described herein.

6. Examples

The present invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes only, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of noncritical parameters which can be changed or modified to yield essentially the same results.

Example 1. Human Genome-Edited Hematopoietic Stem Cells Phenotypically Correct Mucopolysaccharidosis Type I Abstract

Lysosomal enzyme deficiencies comprise a large group of genetic disorders that generally lack effective treatments. A potential treatment approach is to engineer the patient's own hematopoietic system to express high levels of the deficient enzyme, thereby correcting the biochemical defect and halting disease progression. Here, we present an efficient ex vivo genome editing approach using CRISPR-Cas9 that targets the lysosomal enzyme iduronidase to the CCR5 safe harbor locus in human CD34⁺ hematopoietic stem and progenitor cells (HSPCs). The modified cells secrete supra-endogenous enzyme levels, maintain long-term repopulation and multi-lineage differentiation potential, and can improve biochemical and phenotypic abnormalities in an immunocompromised mouse model of Mucopolysaccharidosis type I. These studies provide support for the development of genome-edited CD34⁺ hematopoietic stem and progenitor cells as a potential treatment for Mucopolysaccharidosis type I. The safe harbor approach constitutes a flexible platform for the expression of lysosomal enzymes making it applicable to other lysosomal storage disorders.

Introduction

The present example describes the development of an exemplary in vivo genome editing approach for MPSI. We use CCR5 as the target safe harbor to insert an expression cassette to overexpress IDUA in human CD34⁺ HPSCs and their progeny. CCR5 is considered a non-essential gene because bi-allelic inactivation of CCR5 (CCR5 Δ32) has no general detrimental impact on human health, and the only known phenotypes of CCR5 loss are resistance to HIV-1 infection and increased susceptibility to West Nile virus (19). We report that human HSPCs modified using genome editing to express IDUA from the CCR5 locus engraft and ameliorate biochemical, visceral, musculoskeletal, and neurologic manifestations of the disease in a new immunocompromised model of MSPI.

Results Efficient Targeting of IDUA to the CCR5 Locus in Human HSPCs

To generate human CD34⁺ HPSCs overexpressing IDUA, we used sgRNA/Cas9 ribonucleoprotein (RNP) and adeno-associated viral vector serotype six (AAV6) delivery of the homologous templates (20). RNP complexes consisting of 2′-O-methyl 3′phosphorothioate-modified CCR5 sgRNA (21) and Cas9 protein were electroporated into cord blood-derived (CB) and adult peripheral blood-derived HSPCs (PB). The efficiency of double-strand DNA break (DSB) generation by our CCR5 RNP complex was estimated by measuring the frequency of insertions/deletions (Indel) at the predicted cut site. The mean Indel frequencies were 83%+8 (+SD) in CB-HSPCs and 76%+8 in PB-HSPCs, consistent with a highly active sgRNA. The predominant Indel was a single A/T insertion that abrogated CCR5 protein expression (FIG. 7 ) (22).

To achieve precise genetic modification, the templates for homologous recombination were made by inserting IDUA expression cassettes driven by the spleen focus-forming virus (SFFV) or the phosphoglycerate kinase (PGK) promoter, followed by a yellow fluorescent protein (YFP) downstream of the self-cleaving P2A peptide into the AAV vector genome. A third expression cassette containing IDUA driven by PGK but without a selection marker was also made (FIG. 1A). These strong constitutive promoters were chosen to harness the ability of all hematopoietic lineages to express IDUA and maximize biochemical cross-correction, and because IDUA expression was previously shown not be toxic to HSPCs. Following electroporation, CB and PB cells transduced with the SFFV-IDUA-YFP and PGK-IDUA-YFP viruses were examined for YFP fluorescence to quantify the efficiency of modification. As shown in FIG. 1B, RNP electroporation followed by AAV6 transduction lead to a marked increase in the median fluorescence intensity of the cells. As previously reported, this shift in the fluorescence intensity allows for the identification of cells that have successfully undergone HR-GE18. In CB-derived HSPCs the mean fraction of YFP-positive cells, was 34%±7 and 32%±8 with SFFV and PGK-driven expression cassettes, respectively. In PB-HSPCs, the frequencies were 21%±5, and 24%±5 for the same AAV6 donors (FIG. 1C). AAV6 transduction alone showed <2% YFP-positive cells, while mock cells that underwent electroporation but not AAV transduction had no detectable fluorescence. We measured the efficiency of modification in CB and PB cells transduced with the PGK-IDUA virus lacking the reporter (PGK-IDUA) by genotyping single cell-derived colonies from colony formation assays (CFAs) (FIGS. 8A-8B). In these cells, the frequencies of modification were 54%±10, and 44%±7 in CB and PB-HSPCs, considerably higher than the larger. YFP-containing cassettes, suggesting that efficiency is dependent on insert size (FIG. 1C). Based on these targeting frequencies we conclude that our genome editing protocol is efficient and reproducible for human CB and PB-derived HSPCs.

We also characterized the genomic modifications at CCR5 loci, by quantifying the fraction of targeted alleles in bulk DNA preparations using droplet-digital PCR (ddPCR) (FIGS. 8C, 8D). This data allowed us to estimate the distribution of cells with one (mono-allelic) or two (bi-allelic) alleles targeted in different cell donor samples and indicated that, for the YFP constructs, 65-100% of the cells had mono-allelic modification. Consistent with this, genotyping of YFP-positive colonies in CFAs showed a mean mono-allelic modification frequency of 80%±7.5 (FIG. 1D).

High IDUA Expression in Edited HSPCs and Derived Macrophages

A central concept in our approach is that HSPCs and their progeny will secrete stable, supra-endogenous IDUA levels that can cross-correct the lysosomal defect in affected cells. Examination of modified HSPCs in culture showed that 3 days post-modification, three distinct cell populations could be discerned based on YFP expression: high/medium/low (FIG. 2A). YFP-high cells exhibited persistent fluorescence in culture for at least 30 days, demonstrating stable integration of the cassettes. YFP-negative cells had no detectable YFP expression at the time of selection, though approximately 1% of cells eventually became positive. Most cells with intermediate fluorescence converted to YFP-high (80%) (FIG. 2B). In these cultures, where YFP-positive and negative cells were mixed and grown under expansion conditions, the fraction of YFP-positive cells remained stable for 30 days, suggesting that neither the modification, nor the overexpression of the enzyme, nor reporter expression in vitro impacted the cells' proliferative potential.

When compared to mock-treated cells expressing endogenous IDUA levels, YFP-high cells secreted 250 and 25-fold more enzyme for the SFFV and PGK-driven cassettes respectively, while cell lysates expressed 600 and 50-fold more enzymatic activity (FIG. 2C). Not surprisingly, the SSFV promoter was able to drive substantially higher IDUA expression compared to PGK. When YFP-high IDUA-HSPCs were co-cultured with patient-derived MPSI fibroblasts, they led to a decrease in the average area of lysosomal-associated membrane protein 1 (LAMP-1) positive specks, consistent with reduced lysosomal compartment size and cross-correction of the cellular phenotype (FIG. 2D). These data confirm that IDUA-HSPCs secrete supra-physiological IDUA levels and that the secreted IDUA has the post-translational modifications required for uptake into MPSI cells and cellular cross-correction.

For IDUA-HSPCs to successfully correct biochemical abnormalities in the organs affected in MPSI, they must differentiate into monocytes that will migrate to and differentiate into tissue-resident macrophages such as microglia (brain), Kupffer cells (liver), osteoclasts (bone), and splenic macrophages to deliver the enzyme and cross-correct enzyme-deficient cells. To confirm that IDUA-HSPC could generate macrophages and that these cells can continue to produce IDUA, we differentiated these cells in culture and assayed for IDUA activity. After 3 weeks in a cytokine cocktail containing M-CSF and GM-CSF23, cells had macrophage morphology, expressed the macrophage markers CD11b (˜89%) and CD14 (˜65%), and showed robust phagocytic activity consistent with a macrophage phenotype (FIG. 2E). These IDUA-HPSC-derived macrophages secreted 192-fold and 37-fold more IDUA for the SFFV and PGK-driven cassettes respectively than mock-cell-derived macrophages. Likewise, lysates exhibited 255-fold and 45-fold more IDUA activity (FIG. 2F). These data established that IDUA-HPSC can reconstitute monocyte/macrophages in vitro and that IDUA-HPSC-derived macrophages also exhibit enhanced IDUA expression.

Preserved Repopulation and Differentiation in IDUA-HSPCs

To determine if HSPCs that have undergone genome editing can engraft in vivo, we performed serial engraftment studies into NOD-scid-gamma (NSG) mice. We first tested cells modified with the SFFV and PGK constructs expressing YFP, which allowed us to identify the modified cells in vivo. Equal numbers of CB and PB-derived mock, YFP-negative (YFP−), and YFP-positive (YFP+) cells were transplanted intra-femorally into sub-lethally irradiated 6-8-week-old mice. Primary human engraftment was measured 16 weeks-post-transplantation by establishing the percent of bone marrow (BM) cells expressing both human CD45 and human leukocyte antigens (HLA-ABC) out of total mouse and human CD45⁺ cells (FIG. 3A). For the PGK-driven constructs, the median frequencies of hCD45⁺/HLA+ cells in BM were as follows. Mock 76.25% (min-max: 46.4-95.4%), YFP-21.5% (0.06-89.5%), YFP+4.3% (0.06-96%) (FIG. 3B). This showed a 5-fold drop in repopulation capacity in cells that underwent HR-GE (YFP+) compared to cells that did not but were also exposed to RNP, AAV transduction, and sorting (YFP−). The median frequency of human cells expressing YFP was 0.6% (0-18.5%) and 95.8% (1-100%) for YFP− and YFP+ transplants respectively, confirming that edited cells had engrafted in these mice (FIG. 3C). Human cells were also found in the peripheral blood with median frequencies of 31/3.1/1.1% in mock, YFP−, and YFP+ cells respectively (FIG. 3B).

The apparent engraftment advantage of cells that had not undergone HR-GE was also examined by transplanting bulk populations of HSPCs modified with the cassette without YFP. In two independent experiments, an initial fraction of targeted alleles of 28% (43% modified cells) declined to 5.2% and 6.5% in the engrafted cells (8 and 10% modified cells) despite big differences in human chimerism (FIGS. 3D, 3E). This corresponded to a 5-fold drop in donor 1 and 4-fold drop in donor 2. Interestingly, this fall in targeted alleles showed significant variation in individual mice (2 to 10-fold). This data re-demonstrated the observed loss in engraftment potency after modification (17.18,24,25.26).

Serial transplantation is considered a gold standard to assess self-renewal capacity of HSCs. For secondary transplants, we isolated human CD34⁺ cells from the bone marrow of primary mice and transplanted into secondary mice. YFP+ engrafted mice showed 3.9% (0.8-9.7%) median human cell chimerism, while YFP− mice showed 30.4% (7.7-48.2%) (FIG. 3F). YFP expression in the engrafted human cells was 0.27% (0-1.35) for YFP− cells, and 41.9% (20.8-100) for YFP+ cells (FIG. 3G). Similar levels of human cell chimerism were observed for the SFFV-driven constructs in serial transplants. Collectively, the presence of YFP-expressing cells at 16- and 32-weeks post-modification demonstrates that cells with long-term repopulation potential can be edited, albeit at lower frequencies than cells that did not undergo HR-GE.

To establish the modified cells' ability to differentiate into multiple hematopoietic lineages, we looked in vitro using colony formation unit assays (CFUs) and in vivo after engraftment in NSG mice. In CFUs, CB-derived and PB-derived YFP-expressing cells gave rise to all progenitor cells at the same frequencies as mock-treated and YFP− cells, indicating that IDUA-HSPCs can proliferate and differentiate into multiple lineage progenitors in response to appropriate growth factors. In vivo, B, T and myeloid cells were identified using the human CD19, CD3, and CD33 markers. Compared to mock cells that demonstrated a roughly equal distribution of B and myeloid cells (1:1, CD19:CD33) 16-weeks post-transplantation, YFP+ and YFP− cells showed skewing towards myeloid differentiation (YFP+=1:16, and YFP−=1:5). Examination of the human cell chimerism vs. percent myeloid content per mouse, revealed that low human engraftment is more likely to be associated with a predominant myeloid population. This myeloid bias was not observed in circulating cells in the peripheral blood or in secondary transplants. These data suggest that myeloid skewing is inversely correlated with the degree of human cell engraftment, and that neither the genome editing process, nor IDUA expression, affects the modified cell's capacity to differentiate into multiple hematopoietic lineages in vitro or in vivo.

IDUA-HSPCs Biochemically Correct NSG-IDUAX/X Mice

To determine the potential of human IDUA-HSPCs to correct the metabolic abnormalities in MPSI, we established a new mouse model of the disease capable of engrafting human cells. We used CRISPR-Cas9 to knock-in the W392X mutation, analogous to the W402X mutation commonly found in patients with severe MPSI, into NSG mouse embryos. Homozygous NSG-IDUAX/X mice replicated the phenotype of patients affected with MPSI1 and previously described immunocompetent (27,28) and immunocompromised (29) MPSI mice. We focused the correction experiments on cells expressing IDUA under the PGK promoter, as this promoter has better translational potential because it has decreased enhancer-like activity and less prone to silencing compared to SFFV (30). In the first series of experiments, we examined PB-derived cells in which the modification did not include a selection marker. In bulk transplants, the median human cell chimerism in the bone marrow was 62.2% (min=39.2, max=96.7%) and no statistically significant differences in human engraftment were observed between NSG-IDUAX/X and NSG-IDUAW/X mice (FIG. 4A). GAG urinary excretion was measured at 4, 8, and 18 weeks post-transplantation in NSG-IDUAX/X and IDUAW/X mice. Biochemical correction was detectable after 4 weeks and improved over time (FIG. 4B). This kinetics are consistent with the time lag needed for the genetically engineered HSCs to engraft, expand, and migrate to affected tissues and cross-correct diseased cells. At 18 weeks, NSG-IDUAX/X mice that had been transplanted with IDUA-HSPCs (X/X Tx) excreted 65% less GAGs in the urine compared to sham-treated NSG-IDUAX/X mice (X/X sham) (median Tx=387.2 μg/mg of creatinine, sham=1,122 μg/mg) though the levels had not normalized (W/X sham=155 μg/mg) (FIG. 4B). Transplantation of IDUA-HSPCs also resulted in normalization of tissue GAGs in liver and spleen but not in brain (FIG. 4C). Plasma and brain samples were also analyzed for GAG content and composition by liquid chromatography tandem mass spectrometry (LC-MS/MS) (31). GAGs species including dermatan sulfate, heparan sulfate and keratan sulfate showed statically significant reductions in the plasma but not in the brain of transplanted NSG-IDUAX/X mice. Notably, plasma keratan sulfate in MPSI is derived from bone damage (32) not from decreased IDUA activity suggesting improvement in bone dysplasia in the transplanted mice. Transplantation of IDUA-HSPCs also led to increased IDUA activity to 11.3%, 50.1%, 167.5%, and 6.8% of normal in plasma, liver, spleen, and brain respectively (compared to undetectable in X/X sham) (FIG. 4D). In the spleen, supra-endogenous levels of activity were detected consistently and can be attributed to robust human cell engraftment in this organ in the NSG mouse model. Hepatomegaly also significantly improved.

Because we could not discount the contribution of unmodified cells to the observed correction in bulk transplants, we then examined the effect of HSPCs expressing IDUA and YFP under the PGK promoter after FACS-based selection. Of 15 NSG-IDUAX/X and 5 NSG-IDUAW/X mice, 13/15 and 5/5 were deemed to have engrafted (human chimerism in the bone marrow >0.1%). The median percent human chimerism was 4.2% in heterozygous (median percent YFP+77%) and 9.9% in homozygous mice (median percent YFP+80%) (FIG. 4A). IDUA-YFP-HSCPs increased IDUA tissue activity to 2.9%, 7.4%, 2.5%, and 1.3% of normal in plasma, liver, spleen, and brain respectively (FIG. 4E). Tissue and urine GAGs were also significantly reduced in spleen and liver (FIG. 4F). Together, this data indicates that IDUA-HSPCs can improve the metabolic abnormalities in MPSI and suggest that the degree of correction correlates with human cell chimerism.

IDUA-HSPCs Phenotypically Correct NSG-IDUAX/X Mice

To investigate the effect of IDUA-HSPCs on the skeletal and neurological manifestations of MPSI, sham-treated and transplanted mice also underwent whole body micro-CT and neurobehavioral studies 18 weeks after transplantation. The effect of transplantation on the skeletal system was measured on the skull parietal and zygomatic bone thickness and the cortical thickness and length of femoral bones. In experiments where the mice were transplanted using unselected cells (bulk) and where human cell chimerism was high (FIG. 4A), we observed almost complete normalization of bone parameters by visual inspection and on CT scan measurements (FIGS. 5A, 5B). Mice transplanted with cells that had undergone selection showed partial but statistically significant reduction in the thickness of the zygomatic, parietal bones, and femur (FIG. 5C).

We also examined the open field behavior, passive inhibitory avoidance, and marble-burying behavior of sham-treated and transplanted mice. Transplantation of bulk cells resulted in reduced locomotor activity and long-term memory, regardless of genotype. We suspected that high human-cell chimerism was detrimental for the overall health of the mice. Consistent with this, we observed growth restriction following human cell transplantation in both homozygous and heterozygous mice. This likely represents a toxicity artifact of this xenogeneic transplant model and could be explained in part by the defective erythropoiesis seen in these xenograft models (33). In contrast, NSG-IDUAX/X mice transplanted with YFP-selected cells in which human cell chimerism was not as high exhibited locomotor activity indistinguishable from their sham-treated heterozygous littermates, and markedly higher that the sham-treated knock-out mice (FIG. 5D). These mice also had increased vertical counts at all time points and demonstrated the same exploratory behavior as sham heterozygous mice (FIG. 5E). Transplantation of IDUA-HSPCS in NSG-IDUAX/X also enhanced performance in the passive inhibitory avoidance test 24 h later (FIG. 5F). Digging and marble-burying behavior also improved but did not normalize (FIG. 5G).

Because neuroinflammation has been reported in immunocompetent MPSI mice (34), we looked for effects on brain microgliosis and astrocytosis following transplantation of IDUA-HSPCs. Heterozygous sham-treated (W/X sham), homozygous sham-treated (X/X sham) and homozygous IDUA-HSPC-transplanted (X/X Tx) mouse brains were analyzed 16-week post-transplantation by immunohistochemistry. Microglial activation, assessed by the number of isolectin B4-positive cells (35) was significantly reduced in X/X Tx compared to X/X sham mice (FIG. 5H). Astrocyte activation, as measured by the number of Glial fibrillary acidic protein (GFAP) positive astrocytes, was also significantly reduced (FIG. 5I).

Safety of Our Genome Editing Strategy

To assess genotoxicity and characterize the off-target repertoire of our CCR5 guide, we used the bioinformatics-based tool COSMID (CRISPR Off-target Sites with Mismatches. Insertions, and Deletions) (36). Off-target activity at a total of 67 predicted loci was measured by deep sequencing in two biological replicates of CB-derived HSPCs. In each replicate we compared the percent Indels measured in mock and cells electroporated with RNP with either wild-type (WT) Cas9 or a higher fidelity (HiFi) Cas9 (37). Five of the 67 sites were located within repetitive elements and Indel rates could not be assigned to specific loci in this group. For the remaining 62 genomic locations, sites were deemed true off-targets if. (1) the percent of indels at the site was >0.1% (limit of detection), (2) off-target activity was present in both biological samples, and (3) indels were higher in the RNP compared with the mock samples. Given these criteria only four sites were deemed to be true off-targets (FIG. 6A and Table 1). For all of these sites the frequency of Indels was <0.5% and the use of the HiFi Cas9 abolished off-target activity entirely while maintaining on-target efficiency. Only one exonic site was found in the SUOX gene (sulfite oxidase). The highest off-target activity measured at this site was 0.128%, which was reduced below the limit of detection with HiFi Cas9. These data suggest that our CCR5 sgRNA combined with either WT Cas9 or especially HiFi Cas9 has negligible off-target activity on a large screen of bioinformatically predicted sites.

TABLE 1 Summary of off-target sites (OT′s) above level of detection (>0.1%). PAM sequences  are shown as bold and mismatched bases are shown as italics. For all of these sites the percent of Indels was <0.5% using wild type (WT) Cas9. For 4 of these sites, the use of the HiFi Cas9 abolished off-target activity CCR5 GCAGCATAGTGAGCCCAGAA CCR5 Exon 0.128 93.41 95.832 92.377 93.078 GGG CCR5_ ACAGAATAGAGAGCCCAGAA GRID1 Intergenic 0  0.467  0.434  0.03  0.045 OT3 AGG CCR5_ ACAGCATAGAGGGCCCAGAA SUOX Exon 0  0.105  0.128  0.095  0.051 OT14 GGG CCR5_ ACAGCATAGTGAACCCAGGA TBPL2 Intergenic 0.017  0.388  0.302  0.102  0.052 OT39 GGG CCR5_ GCTGCATAGTGAACCCAGTAT ZNF609 Intergenic 0.032  0.122  0.245  0.031  0.014 OT40 GG

Several studies have shown that in primary cells, Cas9-mediated DSBs result in p53-mediated cell cycle arrest, thereby decreasing the efficiency of HR-GE (38,39). Consequently, concerns have been raised about the potential for enrichment of p53 negative clones when selecting cells that have successfully undergone HR-GE. To examine p53 function in our cells, we targeted four biological samples (two CB and two PB-derived) to express the PGK-IDUA-YFP cassette and separated mock, YFP+ (cells that underwent HR-GE), and YFP− (cells that did not undergo HR-GE). Because p53− clones could be a rare population with a growth advantage, to increase the probability of detection the cells were allowed to expand 100-150-fold (˜2 weeks). We first sequenced all TP53 exons (NM_000546.5) in the three conditions in all four samples using a clinically validated, next-generation sequencing assay. No new TP53 sequence variants were found in the HR-GE+ cells despite in vitro expansion. Consistent with this, after treatment with the DNA double-strand break inducer doxorubicin, mock, HR-GE+, and HR-GE− cells had undisguisable responses when assayed for p53 activation, as measured by p53 protein stabilization by FACs. and transcriptional activation of seven p53 targets genes as measured by qPCR.

Collectively, we performed 200 autopsies (101 mice used in primary engraftment, 50 in secondary engraftment, and 49 in NSG-IDUAX/X correction studies) in which no gross tumors were found. Three tumor-like masses were evaluated by histology and confirmed to be abscesses. These 200 mice were transplanted with a combined dose of 90 million human cells that underwent our genome editing protocol. Considering that the median age for HSCT in MSPI patients is around one year (40), and that an average one year-old is 10 Kg, the total number of modified cells used in this study is roughly equivalent to two clinical doses of 4.5×10⁶ CD34 HSPCs/kg. We conclude that the apparent lack of tumorigenicity and the low off-target activity of the CCR5 sgRNA provide evidence for the safety our modification strategy.

Discussion

We describe an efficient application of RNP and AAV6-mediated template delivery to overexpress IDUA from a safe harbor locus in human CD34⁺ HSPCs. The suitability for CCR5 to be a safe harbor for the insertion and expression of therapeutic genes has been described (30,41). For LSDs like MPSI, the use of the safe harbor would have several advantages compared to genetic correction of the affected locus: (1) it enhances potency, as it allows for supra-endogenous expression, (2) it circumvents design for specific mutations in a gene, (3) the coding sequences can be engineered with enhanced therapeutic properties, e.g., crossing the blood brain barrier (42). (4) it is versatile and easily adaptable to other LSDs, and 5) it avoids the potential risk of uncontrolled integrations (safety).

We studied the self-renewal and multi-lineage differentiation capacity of the modified cells. Our data demonstrates that this approach can modify cells with long-term repopulation potential and preserves multi-lineage differentiation capacity in vivo and in vitro. However, in experiments comparing engraftment potential of the YFP− and YFP+ cells, as well as in bulk transplantation experiments, cells that underwent HR-GE had approximately a 5-fold lower long-term engraftment capacity. Based on observations that HR efficiencies are higher in cycling cells (43,44), one explanation is that HR happens more readily in the cycling progenitor population that in the more quiescent stem cells. The lower engraftment could also represent a negative effect of expression of a foreign fluorescent protein in HSCs (45), as previously substituting a truncated form of the low-affinity nerve growth factor receptor resulted in higher engraftment frequencies than using a fluorescent protein to mark HR-GE cells (18). This is an important caveat for future therapeutic applications, particularly in diseases where high chimerism is required. As observed in allo-HSCT, this engraftment challenge might be partly circumvented by using larger doses of genome-edited cells, which can be facilitated by in vitro expansion in optimized culturing conditions that maintain self-renewal capacity (46,47). Nevertheless, increasing the efficiency of HR-GE in long-term repopulating HSCs will greatly facilitate the clinical application of genome editing in these cells. Current approaches aimed at increasing the efficiency of HR-GE include NHEJ inhibition (48,49,50), HR activation (51,52), limiting p53 pathway activation (53), cell cycle manipulation (44,54), and linking the DNA repair template to the genome editing machinery (50,55).

Ex vivo manipulation of the HSPCs allows for a thorough examination of the genotoxicity and the magnitude of biochemical potency of the cells before delivering the engineered cell product to patients. Through a bioinformatics-guided strategy, we identified four potential off-target sites with minimal off-target activity. Fortunately, all but one, were abrogated by using a higher fidelity nuclease (37). The conclusion that our genome editing strategy is safe is also supported by the lack of tumorigenicity in 200 mice transplanted with 90 million edited cells examined over 16-20 weeks. Furthermore, we showed that HSPCs that have undergone HR-GE and selection had normal p53 function.

Our approach attempts to commandeer the patient's own hematopoietic system to express and deliver lysosomal enzymes and it is based on clinical experience demonstrating superior outcomes in allo-HSCT compared to ERT particularly in the neurological and musculoskeletal symptoms (56,57). The autologous source improves on safety, by eliminating the morbidity of graft rejection, graft-versus-host disease, and immunosuppression, and can lead to earlier intervention by obviating the need for donor matching. Compared to non-targeted gene addition to HPSCs using lentiviruses (7,58), genome editing decreases the potential risks of random viral genome integration and ensures more predictable and consistent transgene expression because the insertion sites are limited to two chromosome loci. Unlike liver-directed approaches using zinc finger nucleases (59), this approach leverages the unique ability of the hematopoietic system to generate tissue macrophages that can migrate into harder-to-treat organs like the CNS (56,57,60,61) and, based on clinical experience with allo-HSCT, will provide better correction in the bone and joints. However, like allo-HSCT, autologous transplantation of genetically modified HSPCs might not be sufficient to abolish all musculoskeletal manifestations in humans (62). While osteoclasts are derived from CD34⁺ cells and might provide enzyme to correct the bone phenotype, chondrocytes are not derived from these cells.

We examined the potential of the edited HSPCs to reverse symptomatology in a new model of MPSI capable of human cell engraftment. Engraftment of the IDUA-HSPCs led to partial enzyme activity reconstitution in plasma and CNS, and normalization in the liver and spleen. Engraftment also resulted in reductions in GAG storage in multiple organs, except the brain. Notably, small changes in circulating and tissue IDUA lead to significant phenotypic improvements. This is not surprising, as even a small fraction of normal IDUA activity can dramatically improve the physical manifestations of MPSI. Mean IDUA activity in fibroblasts from patients with severe MPSI is 0.18% (range 0-0.6), while 0.79% residual activity (range 0.3-1.8) results in mild disease (minimal neurological involvement and the possibility of a normal life span) (63). In fact, healthy individuals can be found with enzymatic activity as low as 4% (64). Our data constitutes the first study to show symptomatic correction of a murine model of an LSD with human genome-edited HSPCs and provides support for the further development of this strategy for the treatment of the visceral, skeletal, and neurological manifestations in MPSI.

Methods AAV Donor Plasmid Construction

The CCR5 donor vectors have been constructed by PCR amplification of ˜500 bp left and right homology arms for the CCR5 locus from human genomic DNA. SFFV, PGK, IDUA sequences were amplified from plasmids. Primers were designed using an online assembly tool (NEBuilder, New England Biolabs, Ipswich, Mass., USA) and were ordered from Integrated DNA Technologies (IDT, San Jose, Calif., USA). Fragments were Gibson-assembled into a the pAAV-MCS plasmid (Agilent Technologies. Santa Clara, Calif., USA).

rAAV Production

We followed a protocol that has been previously reported with slight modifications65. Briefly, HEK 293 cells are transfected with a dual-plasmid transfection system; a single helper plasmid (which contains the AAV rep and cap genes and specific adenovirus helper genes) and the AAV donor vector plasmid containing the ITRs. After 2 days the cells are lysed by three rounds of freeze/thaw, and cell debris is removed by centrifugation. AAV viral particles are purified by ultracentrifugation in iodixanol gradient. Vectors are formulated by dialysis and filter sterilized. Titers are performed using droplet-digital PCR. Alternatively, viruses were amplified and purified by Vigene Biosciences (Rockville, Md., USA).

Electroporation and Transduction of Cells

CCR5 sgRNA was purchased from TriLink BioTechnologies (San Diego, Calif., USA) and was previously reported (22). The sgRNA was chemically modified with three terminal nucleotides at both the 5′ and 3′ ends containing 2′ O-Methyl 3′ phosphorothioate and HPLC-purified. The genomic sgRNA target sequence (with PAM in bold) was: CCR5: 5′-GCAGCATAGTGAGCCCAGAAGGG-3′. Cas9 protein was purchased from Integrated DNA Technologies. RNP was complexed by mixing Cas9 with sgRNA at a molar ratio of 1:2.5 at room temperature. CD34⁺ HSPCs were electroporated 2 days after thawing and expansion by using the Lonza Nucleofector 4D (program DZ-100) in P3 primary cell solution as follows: 10×10⁶ cells/ml, 300 μg/ml Cas9 protein complexed with 150 μg/ml of sgRNA, in 100 μl. Following electroporation, cells were rescued with media at 37° C. after which rAAV6 was added (MOI 15,000 of 15,000 titrated to maximize modification efficiency and cell recovery). A mock-electroporated control was included in most experiments where cells underwent electroporation without Cas9 RNP.

Quantification of Putative CCR5 gRNA Off-Target Activity

Potential off-target sites in the human genome (hg19) were identified and ranked using the recently developed bioinformatics program COSMID (36), allowing up to three base mismatches without insertions or deletions and two base mismatches with either an inserted or deleted base (bulge). The top ranked sites were further investigated. Off-target activity at a total of 67 predicted loci was measured by deep sequencing in two biological replicates of CB-derived HSPCs. Bioinformatically predicted off-target loci were amplified by two rounds of PCR to introduce adaptor and index sequences for the Illumina MiSeq platform. All amplicons were normalized, pooled and quantified using the PerfeCTa NGS quantification kit per manufacturer's instructions (Quantabio, Beverly, Mass., USA). Samples were sequenced on an Illumina MiSeq instrument using 2×250 bp paired end reads. INDELs were quantified as previously described66. Briefly, paired-end reads from MiSeq were filtered by an average Phred quality (Qscore) greater than 20 and merged into a longer single read from each pair with a minimum overlap of 30 nucleotides using Fast Length Adjustment of SHort reads. Alignments to reference sequences were performed using Burrows-Wheeler Aligner for each barcode and the percentages of insertions and deletions containing reads within a ±5-bp window of the predicted cut sites were quantified.

Measuring Insertions at the CCR5 Locus with ddPCR

Genomic DNA was extracted from either bulk or sorted populations using QuickExtract DNA Extraction Solution. For droplet-digital PCR (ddPCR), droplets were generated on a QX200 Droplet Generator (Bio-Rad) per manufacturer's protocol. A HEX reference assay detecting copy number input of the CCRL2 gene was used to quantify the chromosome 3 input. The assay designed to detect insertions at CCR5 consisted of: F:5′-GGG AGG ATT GGG AAG ACA-3′, R:5′-AGG TGT TCA GGA GAA GGA CA-3′, and labeled probe: 5′-FAM/AGC AGG CAT/ZEN/GCT GGG GAT GCG GTG G/3IABkFQ-3′. The reference assay designed to detect the CCRL2 genomic sequence: F:5′-CCT CCT GGC TGA GAA AAA G-3′, R:5′-GCT GTA TGA ATC CAG GTC C-3′, and labeled probe: 5′-HEX/TGT TTC CTC/ZEN/CAG GAT AAG GCA GCT GT/3IABkFQ-3′. The accuracy of this assay was established with genomic DNA from a mono-allelic colony (50% allele fraction) as template. Final concentration of primer and probes was 900 nM and 250 nM respectively. Twenty microliters of the PCR reaction was used for droplet generation, and 40 μL of the droplets was used in the following PCR conditions: 95°—10 min, 45 cycles of 94°—30 s, 57° C.—30 s, and 72°—2 min, finalize with 98°—10 min and 4° C. until droplet analysis. Droplets were analyzed on a QX200 Droplet Reader (Bio-Rad) detecting FAM and HEX positive droplets. Control samples with non-template control, genomic DNA, and mock-treated samples, and 50% modification control were included. Data was analyzed using QuantaSoft (Bio-Rad).

HSPC Selection and Culturing

Human CD34⁺ HSPCs mobilized peripheral blood purchased from AllCells (Alameda, Calif., USA) and thawed per manufacturer's instructions. CD34⁺ HSPCs were purified from umbilical cord blood collected donated under informed consent via the Binns Program for Cord Blood Research at Stanford University and used without freezing. In brief, mononuclear cells were isolated by density gradient centrifugation using Ficoll Paque Plus. Following two platelet washes, HSPCs were labeled and positively selected using the CD34⁺ Microbead Kit Ultrapure (Miltenyi Biotec, San Diego, Calif., USA) per manufacturer's protocol. Enriched cells were stained with APC anti-human CD34 (Clone 561; Biolegend, San Jose, Calif., USA) and sample purity was assessed on an Accuri C6 flow cytometer (BD Biosciences, San Jose, Calif., USA). Cells were cultured at 37° C., 5% CO2, and 5% O2 for 48 hours prior to gene editing. Culture media consisted of StemSpan SFEM II (Stemcell Technologies, Vancouver, Canada) supplemented with SCF (100 ng/ml), TPO (100 ng/ml), FIt3-Ligand (100 ng/ml), IL-6 (100 ng/ml), UM171 (35 nM), and StemRegenin1 (0.75 μM).

Colony Forming Unit Assay and Clonal Genotyping

Cells were single-cell sorted into 96-well plates (Corning) pre-filled with 100 μl of methylcellulose (Methocult, StemCell Technologies).

Single YFP+, YFP−, and mock-treated cells were sorted into methylcellulose media containing SCF, IL3, erythropoietin, and GM-CSF, conditions that support the growth of blood progenitor cells: erythroid progenitors (burst forming unit-erythroid or BFU-E, and colony-forming unit-erythroid or CFU-E), granulocyte-macrophage progenitors (CFU-GM), and multi-potential granulocyte, erythroid, macrophage, megakaryocyte progenitor cells (CFU-GEMM).

After 14 days, colonies were counted and scored as BFU-E, CFU-M, CFU-GM, and CFU-GEMM per the manual for ‘Human Colony-forming Unit (CFU) Assays Using MethoCult’ from StemCell Technologies. For DNA extraction from 96-well plates, PBS was added to wells with colonies, and the contents were mixed and transferred to a U-bottomed 96-well plate. Cells were pelleted by centrifugation at 300×g for 5 min followed by a wash with PBS. Finally, cells were resuspended in 25 μl QuickExtract DNA Extraction Solution (Epicentre, Madison, Wis., USA) and transferred to PCR plates, which were incubated at 65° C. for 10 min followed by 100° C. for 2 min. For CCR5, a 3-primer PCR was set up with a forward primer outside the left homology arm (5′-CACCATGCTTGACCCAGTTT-3′), a forward primer binding the poly-adenylation signal in all inserts (5′-CGCATTGTCTGAGTAGGTGT-3′), and a reverse primer binding inside the right homology arm (5′-AGGTGTTCAGGAGAAGGACA-3′). Accupower premix was used for PCR reaction and cycled at the parameters: 95°—5 min, and 35 cycles of 95°—20 s, 72° C.—60 s. DNA fragments were detected by agarose gel electrophoresis.

Macrophage Differentiation and flow cytometry

CD34⁺ HSPCs were seeded at a density of 2×10⁵ cells/mL in untreated 6-well polystyrene plates in differentiation medium (SFEM II supplemented with SCF (200 ng/ml), Il-3 (10 ng/mL), IL-6 (10 ng/mL), FLT3-L (50 ng/mL), M-CSF (10 ng/ml), GM-CSF (10 ng/ml), penicillin/streptomycin (10 U/mL), and cultured at 37° C. 5% CO2, and 5% 02. After 48 h, non-adherent cells were removed from plates and reseeded in new non-treated 6-well polystyrene plates at 2×10⁵ cells/mL in differentiation medium. Adherent cells were maintained in the same plates in maintenance medium (RPMI supplemented with FBS (10% v/v), M-CSF (10 ng/ml), GM-CSF (10 ng/ml), and penicillin/streptomycin (10 U/mL). After three weeks, adherent cells, comprising terminally differentiated macrophages, were harvested by incubation with 10 mM EDTA and gentle scraping. For phenotypic analysis we harvested 1×10⁵ cells per condition resuspended in 100 μl staining buffer (PBS containing 2% FBS and 0.4% EDTA). Non-specific antibody binding was blocked (5% v/v TruStain FcX, BioLegend, #422302) and cells were stained with 2 μl of each fluorophore-conjugated monoclonal antibody (30 minutes, 4° C., dark). Antibodies used were hCD34-APC (BioLegend #343510), hCD14-BV510 (BioLegend #301842) and hCD11b-PE (BioLegend #101208). Propidium Iodide (1 μg/mL)) was used to detect dead cells and cells were analyzed on a BD FACSAria flow cytometer.

Phagocytosis Assay

pHrodo Red E. coli BioParticles conjugate for Phagocytosis were purchased from ThermoFisher, USA and reconstituted to 1 mg/mL in 10% FBS-containing media. Reconstituted Bioparticles were added to IDUA-HSPC-derived macrophages and incubated at 37° C. for one hour. The cells were then washed and bathed in imaging media (DMEM Fluorobright, 15 mM HEPES, 5% FBS). Imaging followed using the appropriate absorption and fluorescence emission maxima (560 nm and 585 nm, respectively).

Mice

NOD.Cg-PrkdcscidlL2rgtmlWjl/Sz (NSG) mice were developed at The Jackson Laboratory67. Mice were housed in a 12-h dark/light cycle, temperature- and humidity-controlled environment with pressurized individually ventilated caging, sterile bedding, and unlimited access to sterile food and water in the animal barrier facility at Stanford University. All experiments were performed in accordance with National Institutes of Health institutional guidelines and were approved by the University Administrative Panel on Laboratory Animal Care (IACUC 25065).

Transplantation of CD34⁺ HSPCs into NSG Mice

Targeted cells (sorted or bulk) were transplanted four to five days after electroporation/transduction. YFP-negative (YFP−), and YFP-positive (YFP+) cells were isolated using FACS and ˜400.000 cells were transplanted intra-femorally into sub-lethally irradiated (2.1 Gy) 6 to 8-week-old mice. Approximately 1×10⁶ cells HPSCs modified with cassettes without YFP and were transplanted in bulk. Mice were randomly assigned to each experimental group and analyzed in a blinded fashion.

Assessment of Human Engraftment

Sixteen to 18 weeks after transplantation, samples of peripheral blood, bone marrow, and spleen were harvested from recipient mice. Samples were treated with ammonium chloride to eliminate mature erythrocytes. Non-specific antibody binding was blocked (10% vol/vol, TruStain FcX, BioLegend), cells were stained (30 min, 4° C., dark), and analyzed by setting nucleated cell scatter gates using a BD FACSAria II flow cytometer or BD FACSCanto II analyzer (BD Biosciences). Cells were analyzed based on monoclonal anti-human HLA-ABC APC-Cy7 (W6/32, BioLegend), anti-mouse CD45.1 PE-Cy7 (A20, eBioScience, San Diego, Calif., USA), CD19 APC (HIB19, BD511 Biosciences), CD33 PE (WM53, BD Biosciences), anti-mouse mTer119 PE-Cy5 (TER-119, BD Biosciences), and CD3 PerCP/Cy5.5 (HiT3A, BioLegend) antibodies, and Propidium Iodide to detect dead cells. Human engraftment was defined as HLA-ABC+/HCD45⁺ cells. See Supplementary Methods for a complete Antibody list.

IDUA Activity Assay

IDUA enzyme activity was measured fluoremetrically using 4-methylumbelliferyl α-L-iduronide (4MU-iduronide) (LC Scientific Inc., Canada) per established assay conditions (68). Briefly, for IDUA the 4-methylumbelliferyl-iduronide substrate is diluted with sodium formate buffer, 0.4 M, pH 3.5, to 6.6 mM concentration. Twenty-five microliters of aliquots of substrate are mixed with 25 μL of cell or tissue homogenates and adjusted to a final substrate concentration of 2.5 mM. The mixture is incubated at 37° C. for 60 min, and 200 μL glycine carbonate buffer (pH 10.4) is added to quench the reaction. 4-MU (Sigma) is used to make the standard curve. The resulting fluorescence is measured using a SpectraMax M3 plate reader with excitation at 355 nm and emission at 460 nm (Molecular devices).

Analysis of GAGs Using the DMB Method

Urine and tissue GAGs were measured with the modified dimethylmethylene blue assay (DMB)69. Tissue samples (10-30 mg) were incubated for 3 h at 65° C. in papain digest solution (calcium- and magnesium-free PBS containing 1% papain suspension (Sigma), 5 mM cysteine, and 10 mM EDTA, pH 7.4) to a final concentration of 0.05 mg tissue/mL buffer. Fifty microliters of extract was incubated with 200 μL DBM reagent (9:1 31 μM DMB stock (in formiate buffer 55 nM): 2 M Tris base). The samples were read on a microplate reader at 520 nm.

Analysis of GAGs by LC/MS-MS

Disaccharides were produced from polymer GAGs by digestion with chondroitinase B, heparitinase, and keratanase II, resulting in DS (di-0S), HS (diHS-NS, diHS-0S), and KS (mono-sulfated KS, di-sulfated KS). Chondrosine was used as an internal standard (IS). Unsaturated disaccharides, [ΔDiHS-NS, ΔDiHS-0S, ΔDi-4S, mono-sulfated KS and di-sulfated KS were obtained from Seikagaku Corporation (Tokyo, Japan) and used to make standard curves. Stock solutions ΔDiHS-NS (100 μg/ml), ΔDiHS-0S (100 μg/ml), ΔDi-4S (250 μg/ml), mono- and di-sulfated KS (1000 μg/ml) and IS (5 mg/ml) were prepared separately in milliQ water. Standard working solutions of ΔDiHS-NS, ΔDiHS-0S, ΔDi-4S (7.8125, 15.625, 31.25, 62.5, 125, 250, 500, and 1000 ng/ml), and mono- and di-sulfated KS (80, 160, 310, 630, 1250, 2500, 5000, and 10.000 ng/ml) each mixed with IS solution (5 μg/m) were prepared. Mass spectrometer apparatus, run condition, brain homogenate preparation, and disaccharide analysis were done as described in Supplementary Methods.

Histology

After bleeding, brains were trans-cardially perfused with Phosphate-buffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde (PFA) in PBS. Brains were fixed overnight at 4° C. Subsequently, brains were transferred to a 30% sucrose solution overnight for cryoprotection, embedded in Tissue-Tek OCT compound, and cut (15-20 μm sections) on a freezing cryostat (Leica, CM3050). All tissue was stored at −80° C. until further use. For immunohistochemistry, slides were washed in PBS to remove excess OCT. Sections were blocked in 10% normal goat plasma (NGS; Gibco) containing 0.25-3% Triton X-100 for 1 h at 25° C. Primary antibody (anti-GFAP, 1:500) was applied overnight in 10% NGS with 0.1% Triton X-100 at 4° C. followed by the appropriate fluorochrome conjugated secondary antibody (Alexa conjugates; Molecular Probes) for 1 h at 25° C. For imaging microglia, Isolectin GS-IB4 From Griffonia simplicifolia, Alexa Fluor 568 Conjugate (Invitrogen-Molecular Probes, USA) was reconstituted as a 1 mg/ml stock in PBS with 0.5 mM CaCl2 and 0.01% sodium azide and the slides were incubated with a working solution of 5 μg/ml in calcium-containing PBS for one hour at 25° C. Slides were then washed in PBS with 0.1% BSA, counterstained with Hoechst, and mounted in Aqua Poly/Mount (Polysciences, Inc.) for fluorescent microscopy. Slides were visualized by conventional epifluorescence microscopy using an all-in-One Fluorescence Microscope BZ-X800 (Keyence, Itasca. USA).

Immunocytochemistry

MPSI fibroblasts cells (Coriell Cell Repository, GM000798) were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS), blocked with 3% bovine serum albumin (BSA) in PBS, and stained with rabbit anti-LAMP1 (Abcam, ab24170, 1:200) followed by 1:500 dilutions of Alexa 488-conjugated anti-rabbit antibody (Molecular Probes). Mounting and staining of nuclei was done Vectashield with DAPI (Vector labs). Slides were visualized by conventional epifluorescence microcopy using a cooled CCD camera (Hamamatsu) coupled to an inverted Nikon Eclipse Ti microscope. Images were acquired using NIS elements software and analyzed with ImageJ.

Computerized Tomography

High-resolution Micro-CT scans were acquired at Stanford Center for Innovation in In Vivo Imaging (SCI3) using an eXplore CT 120 scanner (TriFoil imaging). Mice were anesthetized with isoflurane (Baxter Corporation, Mississauga, ON, Canada). The scans were obtained with voxel resolution of 100 μm, an energy level of 80 keV, and 360 degrees of whole mice. Microview software (Parallax innovations) was used for isosurface rendering and measurements. Skull thickness was quantified on Midsagittal images. Femur length was determined by measuring the long axis between the two epiphyses. Zygomatic bone thickness was measures on coronal sections, perpendicular to the axis of the zygoma. Bone lengths were determined using the line measurement tool in MicroView. Femurs were measured from the base of the lateral femoral condyle to the tip of the greater trochanter.

Spontaneous Locomotor Activity

All behavioral experimenters were blind to the genotype of the mice throughout testing. All tests were conducted in the light cycle. In all experiments, animals were habituated to the testing room 2 h before the tests and were handled by the experimenter for 3 days before all the behavioral tests. For spontaneous locomotor activity, assessment took place using the open field test in a square arena (76×76 cm2) with opaque white walls, surrounded with privacy blinds to eliminate external room cues. Mice were placed in the center of the open-field arena and allowed to freely move for 10 min while being tracked by Ethovision (Noldus Information Technology, Wageningen, the Netherlands) automated tracking system. Before each trial, the surface of the arena was cleaned with Virkon disinfectant. For analysis, the arena was divided into a central (53.5×53.5 cm2) and a peripheral zone (11.25-cm wide).

Passive Inhibitory Avoidance

The passive inhibitory avoidance test was used to assess fear-based learning and memory. We used a dual-compartment system (GEMINI system, San Diego Instruments), where lighted and dark compartments, equipped with grid floor that can deliver electrical shocks, are separated by an automated gate. On day one, each mouse was habituated to the apparatus by placing it into the lighted compartment. After 30 s, the gate opened allowing access to the dark compartment. When the mice entered the dark compartment, the gate closed and the time to cross after the gate opened is recorded (latency time). On day 2 or training day, the mice receive a 0.5 mA shock for 2 s after a 3 s delay after crossing from the lighted to the dark compartment. On day 3, or testing day, after being placed in the lighted compartment for 5 s, the gate opened allowing access to the dark compartment. The latency to enter the dark compartment was recorded. Maximum time to cross was 10 minutes.

Marble Burying

Repetitive behavior was tested in the marble bury test. Individual mice were introduced into cages containing 20 black glass marbles (1.5 cm diameter, four equidistant rows of five marbles each) on top of bedding 5 cm deep. After 30 min under low-light conditions, mice were removed and the number of marbles that were at least half-covered was determined.

NSG-IDUAX/X Mice

We used CRISPR-Cas9 to knock-in the W401X mutation (UniProtKB—Q8BMG0), analogous to the W402X mutation commonly found in patients with severe MPSI, into NSG mouse embryos. The guide RNA target sequence was searched using crispr.mit.edu and six shortlisted guides close to the target site were first screened by using an in vivo assay in NIH 3T3 cells. Two guides, one each on both sides of the target site, were selected: Guidel (5′-TTATAGATGGAGAACAACTC-3′) cleaves 4 bases upstream and Guide3 (5′-GTTGGACAGCAATCATACAG-3′) cleaves 44 bases downstream of the target site. The guides were prepared by in wtro transcription (HiScribe™ T7 High Yield RNA Synthesis Kit, E2040S. New England Biolabs) of a dsDNA template generated by annealing two oligos (with a 17 promoter in the sense oligo) followed by a standard PCR reaction. The ssODN donor DNA contained an intended point mutation leading to a STOP codon (TGG to TAG): 5′-ggtgggagctagatattagggtaggaagccagatgctaggtatgagagagccaacagcctcagccctctgcttggcttatagATG GAGAACAA/CTCTAGGCAGAGGTCTCAAAGGCTGGGGCTGTGTIGGACAGCAATC ATA/CAGTGGGTGTCCTGGCCAGCACCCATCACCCTGAAGGCTCCGCAGCGGCCT GGAGTAC-3′ (lower case is intron, upper case is exon, guide cut sites marked by “/” and the mutation in bold).

Mouse Zygotes were obtained by mating NSG stud males with super-ovulated NSG females. Female NSG mice 3-4 weeks of age (JAX Laboratories, stock number 005557) were super-ovulated by intraperitoneal injection with 2.5 IU pregnant mare serum gonadotropin (National Hormone & Peptide Program, NIDDK), followed 48 hours later by injection of 2.5 IU human chorionic gonadotropin (hCG, National Hormone & Peptide Program, NIDDK). The animals were sacrificed 14 h following hCG administration and fertilized eggs were collected. CRISPR Injection mixture was prepared by dilution of the components into injection buffer (5 mM Tris, 0.1 mM EDTA, pH 7.5) to obtain the following concentrations: 10 ng/μl Cas9 mRNA (Thermo Fisher Scientific, Carlsbad, Calif.), 10 ng/μl IDUA1F and IDUA3F guide RNA and 10 ng/μl ssODN Donor (Integrated DNA Technologies, Coralville, Iowa). Zygote injections and embryo transfers were performed using standard protocols (70). A total of 38 zygotes were injected, the surviving 27 zygotes were transferred, which yielded seven live offspring. Among these a male homozygous for the mutation was used to establish the NSG-IDUAX/X colony. Mice were genotyped by-PCR based amplification followed by Sanger sequencing using the following primers: GENO F: 5′-CATGGCCCTGTTGGGTGAGTAATGA-3′, and GENO R: 5′-TGTGGTACTCCAGGCCGCTG-3′.

Measurement of p53 Protein Stabilization by FACs

Human HPSCs were incubated in doxorubicin (Sigma) at 0.2 μg/ml for 6 h. After harvesting and washing with PBS, the cells were incubated with LIVE/DEAD fixable blue dead cell stain for 15 min (ThermoFisher, USA). The cells were fixed with 2% paraformaldehyde for 10 min at RT, and permeabilized with 0.1% Triton X-100 in PBS for 10 min at RT. Cells were blocked using 2% goat serum and 0.5% BSA in permeabilization buffer (15 min at RT) and stained with PE-labeled anti-p53 antibody (clone DO-7, Biolegend, USA) or its isotype control for 1 h at RT in the dark. Flow cytometry data was acquired with FACSAria using FACSDiva software (BD Biosciences).

TP53 Gene Sequencing

Sequencing of samples was performed at the Stanford Molecular Genetic Pathology Clinical Laboratory using a clinically validated, targeted next generation sequencing (NGS) assay. Acoustic shearing of isolated genomic DNA (M220 focused ultrasonicator, Covaris, Woburn, Mass.) is followed by preparation of sequencing libraries (KK8232 KAPA LTP Library Preparation Kit Illumina Platforms. KAPABiosystems, Wilmington, Mass.), and hybridization-based target enrichment with custom-designed oligonucleotides (Roche NimbleGen, Madison, Wis.). The panel covers, partially or fully, 164 genes that are clinically relevant in hematolymphoid malignancies, including TP53. Pooled libraries are sequenced on Illumina sequencing instruments (MiSeq or NextSeq 500 Systems, Illumina, San Diego, Calif.). Sequencing results are analyzed with an in-house developed bioinformatics pipeline. Sequence alignment against the human reference genome hg19 is performed with BWA in paired end mode using the BWA-MEM algorithm and standard parameters. Variant calling is performed separately for single nucleotide variants (SNVs), insertions and deletions<20 bp (Indels), and fusions. VarScan v2.3.6 is used for calling SNVs and Indels, and FRACTERA v1.4.4 is used for calling fusions. Variants are annotated using Annovar and Ensembl reference transcripts. The assay can detect variants with a variant allele fraction as low 5%.

qPCR for p53 Target Genes

RNA collection was performed using RNeasy Mini kit (Qiagen, 74104) according to the manufacturer's instructions. RNA (2-7 μg) was treated with DNAse I (Invitrogen DNA-free, AM1906) according to the manufacturer's instructions. Reverse transcription was performed with M-MLV reverse transcriptase (Invitrogen, 28025) and random primers (Invitrogen, 48190). 1 μg of total RNA was used for cDNA synthesis. All samples within an experiment were reverse transcribed at the same time, the resulting cDNA diluted 1:5 in nuclease-free water and stored in aliquots at −80° C. until used. Quantitative PCR was performed in triplicate using PowerUP SYBR green master mix (Life Technologies, A25743) and a 7900HT Fast Real-Time PCR machine (Applied Biosystems). Expression analysis was performed using specific primers for each gene (Table X). The mean of housekeeping gene HPRT was used as an internal control to normalize the variability in expression levels. All qRT-PCR performed using SYBR Green was conducted at 50° C. for 2 min, 95° C. for 10 min, and then 40 cycles of 95° C. for 15 s and 60° C. for 1 min. The specificity of the reaction was verified by melt curve analysis. A standard curve was used to quantify the samples. See Supplementary Methods for the list of qRT-PCR primers.

Statistical Analysis

All statistical test including paired and unpaired t-tests, and one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test was performed using GraphPad Prism version 7 for Mac OS X, GraphPad Software, La Jolla, Calif. USA. Data was reported as means when all conditions passed three normality tests (D'Agostino & Pearson, Shapiro-Wilk, and KS normality test).

REFERENCES

-   1. Wraith, J. E. & Jones, S. Mucopolysaccharidosis type I. Pedia.     Endocrinol. Rev. 12(Suppl 1), 102-106 (2014). -   2. Wraith, J. E. et al. Enzyme replacement therapy for     mucopolysaccharidosis I: a randomized, double-blinded,     placebo-controlled, multinational study of recombinant human     alpha-L-iduronidase (laronidase). J. Pedia. 144, 581-588 (2004). -   3. Mitchell, R. et al. Outcomes of haematopoietic stem cell     transplantation for inherited metabolic disorders: a report from the     Australian and New Zealand Children's Haematology Oncology Group and     the Australasian Bone Marrow Transplant Recipient Registry. Pedia.     Transpl. 17, 582-588 (2013). -   4. Tanaka, A. et al. Long-term efficacy of hematopoietic stem cell     transplantation on brain involvement in patients with     mucopolysaccharidosis type II: a nationwide survey in Japan. Mol.     Genet Metab. 107, 513-520 (2012). -   5. Wynn, R. F. et al. Improved metabolic correction in patients with     lysosomal storage disease treated with hematopoietic stem cell     transplant compared with enzyme replacement therapy. J. Pedia. 154,     609-611 (2009). -   6. Aldenhoven, M. et al. Long-term outcome of Hurler syndrome     patients after hematopoietic cell transplantation: an international     multicenter study. Blood 125, 2164-2172 (2015). -   7. Visigalli, I. et al. Gene therapy augments the efficacy of     hematopoietic cell transplantation and fully corrects     mucopolysaccharidosis type I phenotype in the mouse model. Blood     116, 5130-5139 (2010). -   8. Wang, D. et al. Reprogramming erythroid cells for lysosomal     enzyme production leads to visceral and CNS cross-correction in mice     with Hurler syndrome. Proc. Natl Acad. Sci. USA 106, 19958-19963     (2009). -   9. Biffi, A. et al. Lentiviral hematopoietic stem cell gene therapy     benefits metachromatic leukodystrophy. Science 341, 1233158 (2013). -   10. Eichler, F. et al. Hematopoietic Stem-Cell Gene Therapy for     Cerebral Adrenoleukodystrophy. N. Engl. J. Med. 377, 1630-1638     (2017). -   11. Cartier, N. et al. Hematopoietic stem cell gene therapy with a     lentiviral vector in X-linked adrenoleukodystrophy. Science 326,     818-823 (2009). -   12. McCormack, M. P. & Rabbitts, T. H. Activation of the T-cell     oncogene LMO2 after gene therapy for X-linked severe combined     immunodeficiency. N. Engl. J. Med. 350, 913-922 (2004). -   13. Ranzani, M. et al. Lentiviral vector-based insertional     mutagenesis identifies genes associated with liver cancer. Nat.     Methods 10, 155-161 (2013). -   14. Pan, Y. W., Scarlett, J. M., Luoh, T. T. & Kurre, P. Prolonged     adherence of human immunodeficiency virus-derived vector particles     to hematopoietic target cells leads to secondary transduction in     vitro and in vivo. J. Virol. 81, 639-649 (2007). -   15. Persons, D. A., Hargrove, P. W., Allay, E. R., Hanawa, H. &     Nienhuis, A. W. The degree of phenotypic correction of murine     beta-thalassemia intermedia following lentiviral-mediated transfer     of a human gamma-globin gene is influenced by chromosomal position     effects and vector copy number. Blood 101, 2175-2183 (2003). -   16. Bak. R. O., Gomez-Ospina, N. & Porteus, M. H. Gene editing on     center stage. Trends Genet. 34, 11 (2018). -   17. Schiroli, G. et al. Preclinical modeling highlights the     therapeutic potential of hematopoietic stem cell gene editing for     correction of SCID-XI. Sci. Transl. Med. 9,     https://doi.org/10.1126/scitranslmed.aan0820 (2017). -   18. Dever, D. P. et al. CRISPR/Cas9 beta-globin gene targeting in     human haematopoietic stem cells. Nature 539, 384-389 (2016). -   19. Liu, R. et al. Homozygous defect in HIV-1 coreceptor accounts     for resistance of some multiply-exposed individuals to HIV-1     infection. Cell 86, 367-377 (1996). -   20. Bak, R. O., Dever, D. P. & Porteus, M. H. CRISPR/Cas9 genome     editing in human hematopoietic stem cells. Nat. Protoc. 13, 358-376     (2018). -   21. Hendel, A. et al. Chemically modified guide RNAs enhance     CRISPR-Cas genome editing in human primary cells. Nat. Biotechnol.     33, 985-989 (2015). -   22. Bak, R. O. et al. Multiplexed genetic engineering of human     hematopoietic stem and progenitor cells using CRISPR/Cas9 and AAV6.     Elife 6, https://doi.org/10.7554/eLife.27873 (2017). -   23. Way, K. J. et al. The generation and properties of human     macrophage populations from hemopoietic stem cells. J. Leukoc. Biol.     85, 766-778 (2009). -   24. Wang, J. et al. Homology-driven genome editing in hematopoietic     stem and progenitor cells using ZFN mRNA and AAV6 donors. Nat.     Biotechnol. 33, 1256-1263 (2015). -   25. Genovese, P. et al. Targeted genome editing in human     repopulating haematopoietic stem cells. Nature 510, 235-240 (2014). -   26. De Ravin, S. S. et al. Targeted gene addition in human CD34(+)     hematopoietic cells for correction of X-linked chronic granulomatous     disease. Nat. Biotechnol. 34, 424-429 (2016). -   27. Clarke, L. A. et al. Murine mucopolysaccharidosis type I:     targeted disruption of the murine alpha-L-iduronidase gene. Hum.     Mol. Genet 6, 503-511 (1997). -   28. Wang, D. et al. Characterization of an MPS I-H knock-in mouse     that carries a nonsense mutation analogous to the human IDUA-W402X     mutation. Mol. Genet. Metab. 99, 62-71 (2010). -   29. Mendez, D. C. et al. A novel, long-lived, and highly engraftable     immunodeficient mouse model of mucopolysaccharidosis type I. Mol.     Ther. Methods Clin. Dev. 2, 14068 (2015). -   30. Lombardo, A. et al. Site-specific integration and tailoring of     cassette design for sustainable gene transfer. Nat. Methods 8,     861-869 (2011). -   31. Tomatsu, S. et al. Assay for glycosaminoglycans by tandem mass     spectrometry and its applications. J. Anal. Bioanal. Tech. 2014, 006     (2014). -   32. Fujitsuka, H. et al. Biomarkers in patients with     mucopolysaccharidosis type II and IV. Mol. Genet. Metab. Rep. 19,     100455 (2019). -   33. Hu, Z., Van Rooijen, N. & Yang, Y. G. Macrophages prevent human     red blood cell reconstitution in immunodeficient mice. Blood 118,     5938-5946 (2011). -   34. Wilkinson, F. L. et al. Neuropathology in mouse models of     mucopolysaccharidosis type I, IIIA and IIIB. PLoS ONE 7, e35787     (2012). -   35. Streit, W. J. An improved staining method for rat microglial     cells using the lectin from Griffonia simplicifolia (GSA 1-B4). J.     Histochem. Cytochem. 38, 1683-1686 (1990). -   36. Cradick, T. J., Qiu, P., Lee, C. M., Fine, E. J. & Bao, G.     COSMID: a web-based tool for identifying and validating CRISPR/C as     off-target sites. Mol. Ther. Nucleic Acids 3, e214 (2014). -   37. Vakulskas, C. A. et al. A high-fidelity Cas9 mutant delivered as     a ribonucleoprotein complex enables efficient gene editing in human     hematopoietic stem and progenitor cells. Nat. Med. 24, 1216-1224     (2018). -   38. Ihry, R. J. et al. p53 inhibits CRISPR-Cas9 engineering in human     pluripotent stem cells. Nat. Med. 24, 939-946 (2018). -   39. Haapaniemi, E., Botla, S., Persson, J., Schmierer, B. &     Taipale, J. CRISPR-Cas9 genome editing induces a p53-mediated DNA     damage response. Nat. Med. 24, 927-930 (2018). -   40. Aldenhoven, M. et al. Hematopoietic cell transplantation for     mucopolysaccharidosis patients is safe and effective: results after     implementation of international guidelines. Biol. Blood Marrow     Transpl. 21, 1106-1109 (2015). -   41. Sadelain, M., Papapetrou, E. P. & Bushman, F. D. Safe harbours     for the integration of new DNA in the human genome. Nat. Rev. Cancer     12, 51-58 (2012). -   42. Wang. D. et al. Engineering a lysosomal enzyme with a derivative     of receptor-binding domain of apoE enables delivery across the     blood-brain barrier. Proc. Natl Acad. Sci. USA 110, 2999-3004     (2013). -   43. Heyer, W. D., Ehmsen, K. T. & Liu, J. Regulation of homologous     recombination in eukaryotes. Annu. Rev. Genet. 44, 113-139 (2010). -   44. Yang, D. et al. Enrichment of G2/M cell cycle phase in human     pluripotent stem cells enhances HDR-mediated gene repair with     customizable endonucleases. Sci. Rep. 6, 21264 (2016). -   45. van Galen. P. et al. The unfolded protein response govems     integrity of the haematopoietic stem-cell pool during stress. Nature     510, 268-272 (2014). -   46. Wilkinson, A. C. et al. Long-term cx vivo     haematopoietic-stem-cell expansion allows nonconditioned     transplantation. Nature, doi.org/10.1038/s41586-019-1244-x (2019). -   47. Fares, I. et al. Cord blood expansion. Pyrimidoindole     derivatives are agonists of human hematopoietic stem cell     self-renewal. Science 345, 1509-1512 (2014). -   48. Robert, F., Barbeau, M., Ethier, S., Dostie, J. & Pelletier, J.     Pharmacological inhibition of DNA-PK stimulates Cas9-mediated genome     editing. Genome Med. 7, 93 (2015). -   49. Maruyama, T. et al. Increasing the efficiency of precise genome     editing with CRISPR-Cas9 by inhibition of nonhomologous end joining.     Nat. Biotechnol. 33, 538-542 (2015). -   50. Chu, V. T. et al. Increasing the efficiency of homology-directed     repair for CRISPR-Cas9-induced precise gene editing in mammalian     cells. Nat. Biotechnol. 33, 543-548 (2015). -   51. Yu, S., Song, Z., Luo, J., Dai. Y. & Li. N. Over-expression of     RAD51 or RAD54 but not RAD51/4 enhances extra-chromosomal homologous     recombination in the human sarcoma (HT-1080) cell line. J.     Biotechnol. 154, 21-24 (2011). -   52. Charpentier, M. et al. CtIP fusion to Cas9 enhances transgene     integration by homology-dependent repair. Nat. Commun. 9, 1133     (2018). -   53. Schiroli, G. et al. Precise Gene Editing Preserves Hematopoietic     Stem Cell Function following Transient p53-Mediated DNA Damage     Response. Cell Stem Cell 24, 551-565.e558 (2019). -   54. Lin, S., Staahl, B. T., Alla. R. K. & Doudna. J. A. Enhanced     homology-directed human genome engineering by controlled timing of     CRISPR/Cas9 delivery. Elife 3, e04766 (2014). -   55. Savic, N. et al. Covalent linkage of the DNA repair template to     the CRISPR-Cas9 nuclease enhances homology-directed repair. Elife 7,     https://doi.org/10.7554/eLife.33761 (2018). -   56. Lum, S. H. et al. Long term survival and cardiopulmonary outcome     in children with Hurler syndrome after haematopoietic stem cell     transplantation. J. Inherit. Metab. Dis. 40, 455-460 (2017). -   57. Bjoraker, K. J., Delaney, K., Peters, C., Krivit, W. &     Shapiro, E. G. Long-term outcomes of adaptive functions for children     with mucopolysaccharidosis I (Hurler syndrome) treated with     hematopoietic stem cell transplantation. J. Dev. Behav. Pediatr. 27,     290-296 (2006). -   58. Visigalli, I. et al. Preclinical testing of the safety and     tolerability of LV-mediated above normal alpha-L-iduronidase     expression in murine and human hematopoietic cells using toxicology     and biodistribution GLP studies. Human gene therapy,     doi.org/10.1089/hum.2016.068 (2016). -   59. Sharma, R. et al. In vivo genome editing of the albumin locus as     a platform for protein replacement therapy. Blood 126, 1777-1784     (2015). -   60. Unger, E. R. et al. Male donor-derived cells in the brains of     female sex-mismatched bone marrow transplant recipients: a     Y-chromosome specific in situ hybridization study. J. Neuropathol.     Exp. Neurol. 52, 460-470 (1993). -   61. Shemer, A. et al. Engrafted parenchymal brain macrophages differ     from microglia in transcriptome, chromatin landscape and response to     challenge. Nat. Commun. 9, 5206 (2018). -   62. Schmidt, M. et al. Musculoskeletal manifestations in     mucopolysaccharidosis type I (Hurler syndrome) following     hematopoietic stem cell transplantation. Orphanet J. Rare Dis. 11,     93 (2016). -   63. Oussoren, E. et al. Residual alpha-L-iduronidase activity in     fibroblasts of mild to severe Mucopolysaccharidosis type I patients.     Mol. Genet. Metab. 109, 377-381 (2013). -   64. Elliott, S. et al. Pilot study of newborn screening for six     lysosomal storage diseases using Tandem Mass Spectrometry. Mol.     Genet. Metab. 118, 304-309 (2016). -   65. Khan, I. F., Hirata, R. K. & Russell, D. W. AAV-mediated gene     targeting methods for human cells. Nat. Protoc. 6, 482-501 (2011). -   66. Lee, C. M., Cradick, T. J. & Bao, G. The Neisseria meningitidis     CRISPR-Cas9 system enables specific genome editing in mammalian     cells. Mol. Ther. 24, 645-654 (2016). -   67. Shultz. L. D. et al. Human lymphoid and myeloid cell development     in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human     hemopoietic stem cells. J. Immunol. 174, 6477-6489 (2005). -   68. Zheng, Y. et al. Treatment of the mouse model of     mucopolysaccharidosis I with retrovirally transduced bone marrow.     Mol. Genet. Metab. 79, 233-244 (2003). -   69. de Jong, J. G., Wevers, R. A. & Liebrand-van Sambeek, R.     Measuring urinar glycosaminoglycans in the presence of protein: an     improved screening procedure for mucopolysaccharidoses based on     dimethylmethylene blue. Clin. Chem. 38, 803-807 (1992). -   70. Miura, H., Quadros, R. M., Gunumurthy, C. B. & Ohtsuka, M.     Easi-CRISPR for creating knock-in and conditional knockout mouse     models using long ssDNA donors. Nat. Protoc. 13, 195-215 (2018).

Example 2. Engineering Monocyte/Macrophage Specific Glucocerebrosidase Expression in Human Hematopoietic Stem Cells Using Genome Editing Abstract

Gaucher disease is a lysosomal storage disorder caused by deficiency in the lysosomal enzyme glucocerebroside encoded by the GBA gene. Its hallmark visceral and skeletal manifestations are largely due to pathological organ infiltration and inflammation by diseased macrophages. Intravenous administration of recombinant enzyme and orally-available substrate reduction therapy are currently used to treat it, however, these therapies require life-long administration, and have limited potential to address neuronopathic forms of the disease. An alternative treatment approach is to engineer the patient's own hematopoietic system to restore glucocerebrosidase expression, thereby replacing the affected monocyte/macrophage compartment and potentially constituting a one-time therapy for this disease. We established an efficient ex vivo genome editing approach using CRISPR/Cas9 to target glucocerebrosidase expression cassettes with a monocyte/macrophage-specific element to the CCR5 safe harbor locus in human CD34⁺ hematopoietic stem and progenitor cells. The targeted cells are capable of generating human glucocerebroside-expressing macrophages in vitro and in vivo and maintain long-term repopulation and multi-lineage differentiation potential in serial transplantation studies. This safe-harbor approach using a lineage-specific promoter establishes a universal correction strategy for all pathological mutations in Gaucher disease and circumvents potential detrimental effects of ectopic glucocerebrosidase expression in the stem cell compartment. Furthermore, this approach constitutes a flexible platform to treat other lysosomal enzyme deficiencies.

Here we describe an exemplary embodiment of our generation and characterization of GCase-targeted human HSPCs, an important step towards establishing autologous transplantation of genome-edited cells for GD. We use the RNP/AAV6 platform to achieve efficient integration of GCase cassettes into the CCR5 locus, a proven non-essential locus given the high prevalence of healthy homozygous CCR5 Δ32 individuals in European populations (>10%) (35b) and the observation that homozygous carriers of the Δ32 mutation are resistant to HIV-1 infection (36b). By leveraging a lineage-specific promoter highly expressed in the monocyte/macrophage lineage, we achieve GCase expression in the affected cell lineages while also minimizing ectopic expression in hematopoietic stem and progenitor compartments. GCase-targeted HSPCs demonstrate the capacity for long-term engraftment and multi-lineage differentiation, including the generation of functional macrophages with supraphysiologic GCase expression in vivo.

Result

Efficient targeting of GCase to the CCR5 Locus in Human HSPCs

We used the CRISPR/Cas9 and AAV system to target glucocerebrosidase (GCase) expression cassettes to the human CCR5 safe harbor locus (FIG. 9A). The sgRNA targeting the third exon of CCR5 was previously validated for high on-target activity in primary human HSPCs (29b,37b) and has excellent specificity as prior studies failed to reveal any detectable off-target activity using high-fidelity Cas9 (29b). AAV donor repair templates were generated to drive GCase expression by two different promoters: (1) the Spleen Focus-Forming Virus (SFFV) promoter, which drives constitutive supraphysiologic expression; and (2) the CD68S promoter, a shortened derivative of the endogenous human CD68 promoter with expression restricted to the monocyte/macrophage lineage (38b-40b) (FIG. 9B). This lineage-specific promoter was chosen to minimize potential complications of GCase overexpression in the stem cell compartment. The Citrine-containing vectors were designated SFFV-GCase-P2A-Citrine and CD68S-GCase-P2A-Citrine. A third AAV, CD68S-GCase, lacking the reporter protein, was developed as a more clinically relevant vector for in vivo studies (FIG. 9A).

The targeting efficiencies achievable for each vector were determined by the percent of Citrine-positive (Citrine+) cells and by the percent of CCR5 alleles with on-target cassette integrations using molecular analysis (giving the cell and allele targeting frequencies, respectively). In the presence of both AAV and RNP, the SFFV-driven cassette resulted in approximately 51.5±9.1% (mean±SD) Citrine+ HSPCs 48-hours post-targeting, while AAV alone produced 5.9±4.2% dim Citrine+ cells, likely reflecting episomal expression (FIGS. 9C, 9D). The fraction of CCR5 alleles with on-target cassette integration in the unselected population was 29±9% as measured by droplet digital PCR (ddPCR) (FIG. 9E). To verify targeting in Citrine+ cells, these cells were sorted by FACS and the fraction of modified alleles measured (FIG. 9E). The allelic modification frequency of HSPCs treated with the SFFV-GCase-P2A-Citrine vector that were Citrine+ (SFFV-GCase-Citrine+) was 65.9±4.9%, corresponding to 69% and 31% mono-allelically and bi-allelically targeted cells, respectively. Genotyping of single cell-derived colonies corroborated that 98% percent of the Citrine+ HSPCs were targeted and, consistent with the ddPCR data, showed 67% monoallelic and 33% bi-allelic targeting.

We predicted that because the CD68S promoter should be lineage-specific, Citrine would not be highly expressed in stem and non-myeloid biased progenitor cells and therefore. Citrine expression in HSPCs would not reflect the true editing efficiency of the CD68S-P2A-GCase-Citrine vector (FIG. 9B). Consistent with this, we found that at 48-hours post-modification, Citrine expression from HSPCs treated with the CD68S-GCase-P2A-Citrine AAV and RNP was dim (mean fluorescence intensity (MFI) was 24-fold lower than for the SFFV-GCase-Citrine+ cells) and the mean percentage of CD68S-GCase-Citrine+ HSPCs was 27.7±8.5%, significantly lower than for the SSFV-driven construct despite having comparable CCR5 allele targeting frequencies (32.3±9.6%) (FIGS. 9C-9E). Most importantly, the allele targeting frequency within the CD68S-GCase-Citrine negative population (CD68S-GCase-Citrine-) ranged from 11.8 to 36.4%, confirming the presence of targeted cells lacking Citrine expression (FIG. 9E). We reasoned that the subset of CD68S-GCase-Citrine+ HSPCs likely comprise a subpopulation of granulocyte-monocyte-committed progenitors with increased CD68S promoter activation, while CD68S-GCase-Citrine− HSPCs contain the more primitive populations. Single-cell-derived colony genotyping confirmed that 96.5% of the Citrine+ cells had targeted cassette integrations and showed frequencies of mono-allelic and bi-allelic editing of 64% and 36%, respectively. The allele targeting frequency of the CD68S-GCase vector lacking Citrine was 35.8±7.9% in unselected cell populations corresponding to ˜52% of cells having targeted integrations (FIG. 9E).

Generation of Human GCase-Macrophages from Genome Edited HSPCs

One mechanism by which HSCT is therapeutic in Gaucher disease is through the generation of GCase-expressing macrophages. To confirm the development of macrophages from GCase-targeted HSPCs, we first differentiated control human CD34⁺ HSPCs using a cytokine cocktail including M-CSF, GM-CSF, SCF, IL-3, FLT3 ligand, and IL-641. HSPCs differentiated in this manner exhibited characteristic amoeboid morphology as well as expression of the monocyte/macrophage lineage markers CD14 and CD11b, with concurrent loss of the HSPC marker CD34 (FIGS. 10A, 10B). Following the same differentiation protocol, human HSPCs targeted with the SFFV-GCase-P2A-Citrine and CD68S-GCase-P2A-Citrine constructs, produced macrophages that exhibited Citrine expression, characteristic morphology, and normal phagocytosis of pHrodo-labeled E. coli (FIG. 10C). CD14 and CD11b marker expression in mock-treated, Citrine+ and Citrine− populations from these two constructs revealed comparable expression compared to unmodified cells in all conditions except in CD68S-GCase-Citrine+ cells, which had higher expression in both the standard HSPC and macrophage differentiation conditions (FIGS. 10D and 10E). These results indicate that GCase-targeted HSPCs are capable of producing functional macrophages in vitro and suggest that CD68S-GCase-Citrine+ HSPCs are already primed for differentiation along this lineage.

CCR5 is absent from HSPCs but becomes expressed with monocyte/macrophage differentiation. To examine the effect of our genome editing process on CCR5 expression, we targeted human HSPCs, differentiated them, and quantified CCR5 protein by FACS. In the RNP alone condition, the efficiency of double-strand DNA break generation by our CCR5 RNP complex was estimated by measuring the frequency of insertions/deletions (Indel) at the predicted cut site. The mean indel frequencies in the undifferentiated and differentiated populations was 96.8%±1.2 and 96.4%±1.6 respectively, resulting in almost complete knock-down of CCR5 protein expression). In the presence of both RNP and AAV, cells that successfully underwent HDR (Citrine+) generally lack CCR5 expression, consistent with disruption of both CCR5 alleles by either bi-allelic integration of the cassette or mono-allelic with indel formation in the second allele. In the presence of AAV. CCR5+ cells can be found in the population that did not undergo HDR (˜20%), suggesting that AAV transduction decreases indel generation or exerts a small negative selection in cells containing both AAV and RNP.

The CD68S Promoter Confines GCase Expression to the Monocyte/Macrophage Lineage

The CD68S cassettes were designed to selectively express GCase in the monocyte/macrophage lineage in order to prevent potential toxicity to stem cells from ectopic GCase overexpression. To validate the lineage-specificity of the CD68S promoter, CD68S-GCase-Citrine+ and SFFV-GCase-Citrine+ HSPCs were cultured with growth factors that promoted either HSPC maintenance (HSPC) or macrophage differentiation (MΦ) and Citrine expression was monitored for twenty days. As expected for a constitutive promoter, the fraction of SFFV-GCase-Citrine+ cells remained stable over time in both HSPC and MΦ cultures (>95%). An average of 9.2% and 16.3% of SFFV-GCase-Citrine− cells became positive in the HSPC and MD cultures, respectively, which was consistent with the presence of targeted CCR5 alleles in this population based on ddPCR (FIGS. 11A, 11B). When cultured long-term, the MFI of SFFV-GCase-Citrine+ cells decreased, but the drop in fluorescence intensity was seen exclusively in a subset of cells with very high Citrine expression. Notably, the allele modification frequency did not differ throughout the culturing process, suggesting that the change in Citrine expression was due to regulation of transcription from SFFV promoter or translation but not to selection against the modified cells. In contrast, the percentage of CD68S-GCase-Citrine+ cells decreased in the HSPC cultures but was maintained in the MΦ cultures (FIGS. 11A, 11B). Moreover, there was a substantial increase (˜30-fold) in Citrine MFI from CD68S-GCase-Citrine+ cells in the MΦ compared to the HSPCs culture over the twenty-day differentiation (FIG. 11C).

Because Citrine expression is only a proxy for GCase cassette expression, we also examined GCase protein expression directly by quantifying GCase enzymatic activity in HSPC and MΦ culture conditions. In the HSPC cultures, SFFV-GCase-Citrine+ and CD68S-GCase-Citrine+ cells showed ˜7.7 and 1.3-fold more GCase activity respectively compared to unmodified cells (mock-treated). The CD68S-GCase-Citrine− population showed the same activity as unmodified cells (1.0-fold) supporting the idea that there is no leakage GCase expression from the CD68S promoter in more primitive and non-myeloid HSPCs (FIG. 11D). Macrophages derived from CD68S-GCase-Citrine+ and SFFV-GCase-Citrine+ HSPCs expressed ˜2-fold higher GCase than macrophages derived from mock-treated cells (FIG. 11E). In all but the SFFV-GCase-Citrine+ population, macrophage differentiation resulted in higher levels of GCase expression. This explains the decrease in fold expression in cells targeted with the SFFV-driven cassette with differentiation (from 7.7 to 2.3), as it reflects the marked increase in endogenous GCase (˜4-fold) in the mock cells without a proportional change in exogenous GCase expression from the SFFV expression cassette.

To examine the possibility that differential expression of the GCase cassette was due to changes in the targeted cell populations, we measured the allele targeting frequencies at the time of sorting and post-culture in the HSPC and MΦ cultures using ddPCR (FIG. 11F). We found that the percentage of alleles with on-target cassette integration within Citrine+ and Citrine− populations targeted with both cassettes did not differ between culturing conditions, thus confirming that the changes in expression were attributable to the lineage specificity of the CD68S promoter.

GCase-Targeted HSPCs Sustain Long-Term Hematopoiesis

To examine the potential of GCase-HSPCs to become a one-time therapy for GD1, we tested their long-term repopulation capacity. We first assessed the colony-forming ability of the targeted HSPCs in vitro using the colony-forming unit (CFU) assay. We sorted mock, Citrine+ and Citrine− from SFFV and CD68S targeted populations as single cells in 96-well plates 48-hours post-transplantation and assessed their phenotype 14 days later. Notably, SFFV-GCase-Citrine+ HSPCs produced the fewest colonies of all conditions and exhibited the highest variability in the distribution of colony phenotypes formed, suggesting that supraphysiologic GCase expression or other aspects of SFFV promoter physiology may have a toxic effect on HSPCs (FIG. 12A). As predicted by the model of restricted lineage expression of the CD68S promoter, CD68S-GCase-Citrine+ HSPCs formed exclusively CFU-GM's (granulocyte/monocyte), while the cells that did not express Citrine (CD68S-GCase-Citrine-) produced a normal distribution of colony phenotypes (FIG. 11B). These results strongly support our previous hypothesis that CD68S-GCase-Citrine+ cells in undifferentiated HSPCs represent granulocyte/monocyte primed progenitors and that bona fide CD68S-GCase-P2A-Citrine-targeted stem cells reside within the CD68S-GCase-Citrine-population.

To test in vivo engraftment potential, GCase-targeted HSPCs were serially transplanted into NOD.Cg-PrkdcscidlL2rgtmlWjl/Sz (NSG) mice. Cell doses varied from 2.5×10⁵ to 2×10⁶ HSPCs and were dependent on the CD34⁺ cell yield per human donor. We focused our long-term engraftment experiments on the CD68S-GCase-P2A-Citrine and CD68S-GCase vectors because of the potential detrimental effect of the SFFV promoter, its observed drop in expression, and its barriers to clinical translation. Targeted cells were transplanted without selection intrafemorally or intrahepaticaly into sublethally irradiated NSG mice. Primary human engraftment was quantified after sixteen weeks as the percentage of cells expressing human CD45 within the total hematopoietic population (mouse CD45⁺ and human CD45⁺).

Transplantation of GCase-targeted HSPCs resulted in substantial human cell chimerism. In the bone marrow, the median human cell chimerism was 23.2% (min: 0.17%; max: 91.5%) and 50.6% (0.53%; 91.7%) in CD68S-GCase-targeted and CD68S-GCase-P2A-Citrine-targeted cells, respectively (FIG. 12C). Similar engraftment numbers were seen in the spleen: 20.4% (0.14%, 79.3%) for the cassette lacking Citrine and 35.8% (0.38%; 89.6%) for the cassette containing Citrine (FIG. 12D). To determine the proportion of engrafted cells derived from targeted HSPCs, the targeted allele frequency of the engrafted hCD45⁺ population in the bone marrow was measured using ddPCR in cell preparations that included mouse and human CD45⁺ cells as the ddPCR assay recognizes only human alleles (FIG. 12E). The median allele targeting frequencies of the engrafted cell populations were 4.4% (min: 0.23%: max: 51.0%) and 4.2% (0.73%; 34.6%) for the CD68S-GCase and CD68S-GCase-P2A-Citrine cassettes, respectively; however, allele targeting frequency varied highly across human cell donors and mice. The allele targeting frequency of the engrafted cells tended to be lower compared to the transplanted HSPCs, with an observed drop ranging from 1.9 to 12.5-fold. Because cell doses of transplantation varied in the mice targeted with the Citrine-containing construct, the mice were colored-coded and tracked for engraftment and targeting efficiency in engrafted cells. This suggested a correlation between higher cell dose and higher engraftment of modified cells, a finding that is not surprising as there are likely more targeted long-term stem cells available for engraftment.

Serial engraftment studies are the gold standard to determine self-renewal capacity of hematopoietic stem cells. Secondary transplants were performed by isolating human CD34⁺ cells from bone marrow in eight 16-week mice (7 from CD68S-GCase and one from CD68S-GCase-P2A-Citrine targeted cells) and transplanting them (without pooling) into eight NSG recipient mice. Human engraftment and allele targeting frequency were assessed 16 weeks later (32 weeks post-modification) as previously described. The median human cell chimerism of all transplants was 10% (Range: 0.04%-48.9%) (FIG. 12F). Droplet digital PCR analysis of the engrafted cells from mice with human cell chimerism >1% (n=5) showed a median allele targeting frequency of 21.9% (min: 1.3%; max: 40.5%), compared to 6.3% in the cells prior to transplantation (FIG. 4G). We reason that this increase in allelic targeting pre-to-post transplantation in secondary transplants reflects that targeted HSPCs that undergo primary engraftment in an NSG recipient have high engraftment potential and confirms the presence of long-term repopulating hematopoietic stem cells in the genome-edited population that are capable of long-term engraftment in vivo.

In Vivo Monocyte Macrophage Lineage Differentiation of GCase-Targeted HSPCs

To examine the multi-lineage differentiation potential of GCase-targeted HSPCs in vivo we measured lymphoid and myeloid engraftment by the expression of the cell surface markers hCD19 (B-cells) and hCD33 (pan-myeloid), respectively. We included only mice with human engraftment greater than 1% as these have sufficient cell numbers to reliably measure myeloid and lymphoid reconstitution. In primary engraftment studies, the median percentage of myeloid cells and B-cells in the bone marrow was 27.4% and 65.9%, respectively, for the mice transplanted with CD68S-GCase-targeted HSPCs, and 19.3% and 70%, respectively, for the mice transplanted with CD68S-GCase-P2A-Citrine-targeted HSPCs (FIG. 13A). In general, B-cell production was higher than myeloid and consistent with what has been previously reported for unmodified cells (42b,43b). We similarly found myeloid and lymphoid cell production in secondary engraftment mice in 5 of the 8 mice with bone marrow chimerism greater than 1% (FIG. 13B).

To assess the lineage-specificity of the CD68S promoter in vivo, we compared Citrine expression in the B-lymphoid and myeloid compartments in primary engraftments studies of CD68S-GCase-P2A-Citrine-targeted HSPCs that had robust engraftment of targeted cells (allele modification fraction >10%). As expected, expression of the CD68S-GBA-P2A-Citrine cassette was restricted to the myeloid (CD33⁺) and monocyte lineages (CD14⁺), with more frequent expression observed in monocytes (FIGS. 13C and 13D). Despite robust modification in the bone marrow, three mice did not show Citrine expression in monocytes, which could be due to incomplete differentiation along this lineage since the human cells are lacking the appropriate cytokines or expression that is below our rigorous gating strategy. Because the generation of GCase-expressing macrophages is critical to addressing Gaucher disease pathophysiology, it was also important to verify that engrafted, GCase-targeted HSPCs have the capacity to produce human macrophages with heterologous GCase expression. Towards this end, human CD14⁺ monocytes were isolated via FACS from the bone marrow of transplanted mice 16 weeks post-transplantation and differentiated by adding human macrophage colony stimulating factor (M-CSF). This step was performed in vitro because mouse M-CSF, a cytokine required for macrophage differentiation, does not have activity on human cells (44b). Human macrophages differentiated in this manner showed expression of the lineage marker CD68 as well as Citrine (12.3±4.5% of human CD68+ cells), verifying that engrafted, targeted HSPCs can produce macrophages that express the therapeutic GCase cassette (FIG. 13E).

To improve engraftment and differentiation of myeloid lineages of our modified HSPCs in vivo, we performed transplantation experiments in NSG-SGM3 mice. These are NSG mice expressing human interleukin-3 (IL-3), human granulocyte/macrophage-stimulating factor (GM-CSF), and human Stem Cell Factor (SCF or KIT-ligand), cytokines that support the engraftment and differentiation of human myeloid lineages (45,46). At 16-weeks, transplantation of CD68S-GCase-P2A-Citrine-targeted cells resulted in median human cell chimerism of 17.7% (min: 5.1%; max: 39.6%), 61.7% (min: 22.1%; max: 85.8%), and 33.6% (min: 1.8%; max: 72%) in the bone marrow, spleen, and peripheral blood respectively (FIG. 14A). The median allele targeting frequencies of the engrafted cell populations were 15.6% (min: 12%; max: 20%), 20.4% (min: 16%; max: 25%), 5.0% (min: 2%; max: 29%) in the same tissues (FIG. 14B). The observed drop in modified engrafted cells relative to the pre-transplant level (43%) was 2.7-fold in the bone marrow, consistent with but in the low range of studies in NSG mice (FIG. 12E). We observed B, myeloid, and monocyte development with less preponderance of B-lymphoid population compared to NSG mice. As before, Citrine+ cells were seen exclusively in the myeloid and monocyte cells (FIG. 14C). Tissue macrophages were extracted from liver and lung using an enzymatic method and peritoneal macrophages were obtained by analysis of peritoneal fluid. We found robust human cell populations that were CD45⁺ or CD45/CD11b+ as well as Citrine+ in these macrophage cell preparations (FIGS. 14D-14F). Samples with high cell numbers that allowed enrichment of live human-myeloid-Citrine+ for enzymatic analysis were sorted and the GCase activity measured. Consistent with our studies of HSPCs differentiated in culture, the Citrine+ cells expressed 2.0 (bone marrow), 2.1 (spleen), and 1.6-fold (lungs) higher GCase than Citrine− cells (FIGS. 11E and 14G). Analysis of targeted CCR5 alleles from sorted cells populations including bone marrow, lung, spleen, liver, and peritoneal macrophages show enrichment of targeted alleles in the Citrine+ cells compared to Citrine-cells, confirming that the observed Citrine expression is from targeted cells (FIG. 14H).

Discussion

Gaucher disease is currently treated using enzyme replacement therapy (ERT) and substrate reduction therapy (SRT). Both approaches have been shown to be effective at addressing hematological and visceral manifestations (9-13) and can reduce, but not eliminate, bone complications in this disease (47-49). Neither ERT, not the best tolerated form of SRT (eliglustat), are expected to impact neuronopathic forms of GD (GD2 and GD3) or the increasingly recognized neurological symptoms in GD1 (50,51). ERT involves life-long, bi-weekly infusions, and the development of antibodies can, in some cases, decrease enzyme bioavailability and impact clinical outcome (52-56). Approved SRTs (miglustat and eliglustat) also require life-long administration, repeated dosing (three and two times per day respectively) and, particularly for miglustat, significant side effects due to nonspecific inhibition of other enzymes (57). Both modalities are very costly with estimated annual cost of $300,000 to $450,000 (estimated life-time cost of ˜$6 to $22 million) limiting their availability worldwide (58-60). In the past, allo-HSCT was used effectively and led to rapid improvement in the hematological and visceral parameters as well as regression of skeletal disease, but given its significant morbidity and mortality, its use has been reserved for individuals with neurologic or progressive disease unresponsive to ERT and SRT (61-65). Specifically, allo-HSCT has shown potential to halt neurological progression in patients with GD type 3 (D3) when treated at young age and early in the disease process (66-69).

Given the potential for HSCT to constitute a one-time therapy for GD1 and its likely beneficial effect in the CNS, improving the safety of HSCT for GD would be a significant development. The use of autologous HSPCs is safer because it eliminates the morbidity of graft-versus-host disease, results in faster engraftment, and can lead to earlier intervention by obviating the need for donor matching. For this reason, non-targeted lentiviral-mediated delivery of constitutively expressed GCase is being explored in HSPCs and has yielded promising results in murine GD models where transplantation of these cells achieved normalization of GCase levels, reduced Gaucher cell infiltration, and lowered glucocerebroside storage (21b-23b). However, because of the pseudorandom integration of the viral genomes, concerns remain about its potential for tumorigenicity (24b,25b). Genome editing, as a more precise genetic tool, decreases the chance of random integration and ensures more predictable and consistent transgene expression. In addition to the hematopoietic system, the liver has also been considered as potential enzyme replacement depot and in vivo liver-directed approaches using zinc finger nucleases have also been investigated in mouse models (70b). However, it is not clear the liver-secreted GCase would have the proper glycosylation to cross-correct affected cells or that it could cross into the CNS. Transplantation of ex vivo genome-edited HSPCs can provide direct replacement of pathological cells and leverages the ability of graft-derived macrophages that can migrate to the brain (19b) and bone. Therefore, autologous transplantation of gene-corrected cells, if coupled with safer conditioning regimens, could be a promising therapy for GD patients regardless of disease subtype.

To begin the development of autologous transplantation of genome-edited hematopoietic stem cells, we established an efficient application of CRISPR/Cas9 to target a functional copy of GCase into human CD34⁺ HSPCs. Here, we use sgRNA/Cas9 and AAV6-mediated template delivery to target GCase to the CCR5 locus, a gene previously used for the insertion and expression of therapeutic genes (33b,34b). CCR5 is considered a safe harbor because germline deletions in this gene are common (up to 10% in the Northern European population) and have no overt developmental phenotype (35b). Germline CCR5 loss might be beneficial as it provides protection against HIV36, and possibly smallpox (71b), although it also appears to reduce protection against influenza (72b) and West Nile virus (73b). Compared to genetic correction of the affected locus, the use of a safe harbor constitutes a universal therapy for all patient mutations and has greater designability as regulatory and GCase protein sequences can be engineered with enhanced therapeutic properties. For targeting Gaucher disease specifically, it circumvents the design of genetic tools for the GBA locus, which can be non-specific given the presence of GBAP, a pseudogene with 96% sequence homology to the GBA gene (74b).

To express GCase from the CCR5 locus, we used a previously characterized derivative of the CD68 promoter and confirmed through in vitro and in vivo differentiation protocols that it achieves monocyte/macrophage specific expression of GCase (38b-40b). We reasoned that because the primarv manifestations of Gaucher disease are due to pathology in monocyte/macrophage lineage cells, enzyme reconstitution in this lineage should be sufficient to provide phenotypic correction in this disease. Furthermore, our studies with the SFFV promoter did not consistently result in sustained GCase and reporter expression in human HSPCs, suggesting that high and sustained GCase in the stem and progenitor compartment might have detrimental effects. This would not be surprising, as negative impact in long-term engraftment by lysosomal enzyme overexpression has been seen previously for galactocerebrosidase (75b). Furthermore, transplantation using retrovirally transduced CD34⁺ HSPCs in human where GCase was driven by the LTR promoter failed to show long-term reconstitution (18b). While several reasons can explain this observation, including cell dose and lack of conditioning, one explanation is that constitutive GCase expression by the LTR had a detrimental effect in the repopulating stem cell.

We examined the ability of the targeted human HSPCs to engraft and differentiate in serial transplantation studies in immunocompromised mice and demonstrate that our approach can modify cells with long-term repopulation potential and preserves multi-lineage differentiation capacity. We re-demonstrated a reduced repopulation capacity of the edited HSPC population in primary engraftment studies reported previously for engineered HSPCs in viral-mediated gene addition and gene-editing contexts (29b,30b,76b). However, the enhanced allele modification frequencies in the secondary transplants suggest that this initial decreased capacity is due to a reduced number of targeted long-term repopulating stem cells (LT-HSCs) compared to targeted shorter-lived progenitors and not to detrimental effect on engraftment per se. Interestingly, the allele targeting frequency of the engrafted cell population increased in some cases suggesting that the variability in targeted HSPC engraftment may be accounted for by stochastic engraftment dynamics driven by oligoclonal reconstitution (77b). Even though these experiments do not achieve 100% human cell chimerism, transplantation outcomes in humans and mice indicate that low level chimerism could be sufficient to provide symptomatic relief (78b,79b). Specifically, in mice, 7% wildtype cell engraftment was shown to be sufficient to reverse disease pathology (80b). In our primary engraftment studies, the median allele modification frequency of the engrafted cells was ˜4%, which corresponds to 4-8% of targeted cells (depending on the ratio biallelic or monoallelic modification in the engrafted cells) and an 8-16% unmodified cell dose (given that our cells express 2-fold more GCase). Future experiments in the appropriate immunocompromised models of GD to allow engraftment and proliferation of human cells will establish the potential of these cells to correct the phenotype. Regardless of the outcome, future efforts aimed at increasing the permissiveness of long-term HSCs to undergo homology-dependent genome editing will be important for the therapeutic application of these cells.

Herein, we report the use of a genome editing to target a safe harbor to create lineage-specific expression of proteins. This approach is highly flexible and could serve as a platform to restore the expression of lysosomal enzymes and potentially other secreted proteins with therapeutic potential, provided the therapeutic cassettes are within the packaging capacity of AAV. These studies exemplify a specific use for this approach for the expression of human glucocerebrosidase as a potential intervention for the definitive treatment of GD and support further preclinical development of this strategy.

Methods AAV Vector Plasmid Construction

The CCR5 donor vectors have been constructed by PCR amplification of ˜500 bp left and right homology arms for the CCR5 locus from human genomic DNA. SFFV and GBA sequences were amplified from plasmids. The CD68S sequence was obtained from Dahl et al, 201581 and was cloned from a gblock Gene Fragment (IDT, San Jose, Calif., USA). Primers were designed using an online assembly tool (NEBuilder, New England Biolabs, Ipswich, Mass., USA) and were ordered from Integrated DNA Technologies (IDT, San Jose, Calif., USA). Fragments were Gibson-assembled into a the pAAV-MCS plasmid (Agilent Technologies, Santa Clara, Calif., USA).

rAAV Production

rAAV was produced using a dual-plasmid system as previously described82. Briefly. HEK293 cells were transfected with plasmids encoding an AAV vector and AAV rep and cap genes. HEK293 cells were harvested 48-hours post-transfection and lysed using three cycles of freeze-thaw. Cellular debris was pelleted by centrifugation at 1350 g for 20 minutes and the supernatant collected. Active rAAV particles were purified using iodixanol density gradient ultracentrifugation, dialyzed in PBS, and stored in PBS at −80° C. rAAV vectors for in vivo applications was ordered from Vigene Biosciences (Rockville, Md., USA). Viral titers were determined using droplet digital PCR with the following primer/probe combination: F: GGA ACC CCT AGT GAT GGA GTT, R: CGG CCT CAG TGA GCG A, P: /56FAM/CAC TCC CTC/ZEN/TCT GCG CGC TCG/3IABkFQ/.

HSPC Isolation and Culturing

Human CD34⁺ HSPCs mobilized from peripheral blood were purchased frozen from AllCells (Alameda, Calif., USA) and thawed per manufacturer's instructions. Cord-blood derived human CD34⁺ HSPCs were obtained through the Binns Program for Cord Blood Research at Stanford University. Briefly, mononuclear cells were isolated by density gradient centrifugation using Ficoll Plaque Plus density gradient medium followed by two platelets washes. CD34⁺ mononuclear cells were positively selected using CD34⁺ Microbead Kit Ultrapure (Miltenyi Biotec, San Diego, Calif., USA) per manufacturer's instructions. Purity of the isolation was assessed by staining cells with APC-conjugated anti-human CD34⁺ (Clone 561; Biolegend, San Jose, Calif., USA) and analyzing the fraction of APC⁺ cells using an Accuri C6 flow cytometer (BD Biosciences, San Jose, Calif., USA). Cells were cultured in media consisting of StemSpan SFEM II (Stemcell Technologies, Vancouver, Canada) supplemented with SCF (100 ng/ml), TPO (100 ng/ml), Flt3-Ligand (100 ng/ml), IL-6 (100 ng/ml), UM171 (35 nM), and StemRegenin1 (0.75 mM).

Gene Editing in HSPCs

An sgRNA targeting CCR5 exon 3 (sequence: 5′-GCAGCATAGTGAGCCCAGAA-3′) was purchased from TriLink Biotechnologies (San Diego, Calif., USA) with the chemical modification 2′-O-methyl-3′-phosphorothioate (31b). Cas9 and Hifi Cas9 were purchased from Integrated DNA Technologies (IDT, San Jose, Calif., USA Catalog #1081058 and #1081060). The editing procedure was performed as follows: sgRNA and Cas9 protein were complexed at a molar ration of 1:2.5 (sgRNA:Cas9) at room temperature for 5 minutes. The RNP was electroporated into human CD34⁺ HSPCs 48 hours after thawing using the Lonza 4D nucleofector with the following conditions: pulse code: DZ100; cell density: 1×10⁶ cells in 100 μl; [Cas9]: 30 μg; [sgRNA]: 15 ug. Following electroporation, cells were immediately rescued with HSPC culture media pre-warmed to 37° C. rAAV6 was applied to cells at an MOI of 10,000-20,000.

Measurement of Cassette Integration Using ddPCR

Genomic DNA was extracted from selected or unselected cell populations using QuickExtract DNA Extract Solution and digested using AFIII (New England Biosciences). Two detection probes were used in the assay to simultaneously quantify wildtype CCLR2 reference alleles gene-targeted CCR5 alleles. The ratio of detected CCLR2/CCR5 events gave the fraction of targeted alleles in the original cell population. The CCR5 detection assay was designed as follows: F:5′-GGG AGG ATT GGG AAG ACA-3′, R: 5′-AGG TGT TCA GGA GAA GGA CA-3′, labeled probe: 5′-FAM/AGC AGG CAT/ZEN/GCT GGG GAT GCG GTG G/3IABkFQ-3′. The reference assay was designed as follows: F:5′-CCT CCT GGC TGA GAA AAA G-3′, R: 5′-GCT GTA TGA ACT CAG GTC C/3IABkFQ-3′. Primer and probes final concentrations were 900 nM and 250 nM, respectively. 20 μL of the PCR reaction was used for droplet generation. 40 μL of droplets was used in a PCR reaction with the conditions: 95° C. for 10 min, 45 cycles of melting at 94° C. for 30 s, annealing at 57° C. for 30 s, and extension at 72° C. for 2 min, with a final extension at 98° C. for 10 min. All steps were performed with ramping of 2 C/s and reactions were stored at 4° C. covered from light until droplet analysis. Analysis was performed on a Qx200 Droplet Reader (Bio-Rad) detecting FAM and HEX positive droplets. Control samples included Mock (non-modified) genomic DNA and no-template control. Data analysis was performed using Quantasoft (Bio-Rad).

Colony-Forming Unit Assay and Clonal Genotyping

Colony-Forming Unit assays were performed using Methocult methylcellulose (StemCell Technologies) as per the manufacturer's protocol. Briefly, CD34⁺ HSPCs were single-sorted into 96-well flat-bottom plates (Corning) pre-filled with 100 ul Methocult. Cells were cultured for fourteen days at 37° C., 5% O and 5% CO2. Colonies were quantified and characterized morphologically by color, size and shape as burst-forming unit—erythroid (E-BFU), colony-forming unit—erythroid (E-CFU), colony-forming unit—granulocyte/monocyte (CFU-GM) or colony-forming unit—granulocyte/erythroid/macrophage/megakaryocyte. Colonies were genotyped by extracting genomic DNA in QuickExtract DNA Extraction Reagent (Lucigen, QE09050) and performing a 3-primer in-and-out PCR to amplify both wild-type CCR5 alleles and CCR5 alleles with targeted integrations. The 3-primer in-and-out PCR utilized a forward primer out the left CCR5 homology arm (5′-CACCATGCTTGACCCAGTTT-3′), a forward primer binding the poly-adenylation signal in the cassette (5′-CGCATTGTCTGAGTAGGTGT-3′), and a reverse primer binding inside the right homology arm (5-AGGTGTTCAGGAGAAGGACA-3′). Accupower pre-mix (Bioneer, Oakland, Calif.) was used for the PCR with cycling parameters: 95° C. for 5 min, and 35 cycles of 95° C. for 20 s, 72° C. for 60 seconds. DNA fragments were detected by agarose gel electrophoresis. Wild-type and targeted CCR5 alleles yielded bands of 590 base-pairs and 1100 base-pairs, respectively.

Macrophage Differentiation and Flow Cytometry

CD34+ HSPCs were seeded at a density of 2×105 cells/mL in non-treated 6-well plates in differentiation medium (SFEM 11 supplemented with SCF (200 ng/ml), 11-3 (10 ng/mL), IL-6 (10 ng/mL). FLT3-L (50 ng/mL), M-CSF (10 ng/ml) and penicillin/streptomycin (10 U/ml)). After 48 hours, non-adherent cells were removed and reseeded in a new non-treated 6-well plate at 2×105 cells/mL in differentiation medium. Adherent cells were maintained in the same dish in maintenance medium (RPMI supplemented with FBS (10% v/v), M-CSF (10 ng/ml) and penicillin/streptomycin (10 U/ml)). After two weeks, adherent macrophages were harvested by incubation with 10 mM EDTA in PBS. For phenotypic analysis, 1×105 cells per condition were harvested and resuspended in 100 μl staining buffer comprised of PBS supplemented with 2% FBS and 0.4% EDTA. Non-specific antibody binding was blocked (5% v/v TruStain FcX, BioLegend, #422302) and cells were stained with 2 μl of each fluorophore-conjugated monoclonal antibody (30 minutes, 4° C., dark). Antibodies used were hCD34-APC (BioLegend #343510), hCD14-BV510 (BioLegend #301842) and hCD11b-PE (BioLegend #101208). Propidium Iodide (1 μg/mL) was used to detect dead cells and cells were analyzed on a BD FACSAria flow cytometer.

Phagocytosis Assay

pHrodo Red E. coli BioParticles conjugate for Phagocytosis were purchased from ThermoFisher, USA and reconstituted to 1 mg/mL in 10% FBS-containing media. Reconstituted Bioparticles were added at a final concentration of 0.1 mg/mL to IDUA-HSPC-derived macrophages and incubated at 37° C. for one hour. The cells were then washed and bathed in imaging media (DMEM Fluorobright, 15 mM HEPES, 5% FBS). Imaging followed using the appropriate absorption and fluorescence emission maxima (560 nm and 585 nm, respectively) with a BZ-X710 Keyence fluorescence microscope.

Transplantation of CD34⁺ HSPCs into NSG Mice

Targeted HSPCs (unselected) were transplanted 48 hours post-targeting into sub-lethally irradiated NSG recipients. Primary transplants were performed by intrahepatic injection into newborn pups or by intrafemoral injection at 6-8 weeks of age. Approximately 1×106 cells were transplanted into each mouse for all primary transplants. For secondary transplants, human CD34⁺ HSPCs were isolated from transplanted 16-week old mice at the time of primary engraftment analysis using CD34⁺ Microbead Kit Ultrapure (Miltenyi Biotec, San Diego, Calif., USA) and transplanted without pooling into a second sub-lethally irradiated NSG recipient. Secondary transplants were performed by intrahepatic injection into newborn pups.

Assessment of Human Cell Engraftment

16 weeks post-transplantation, peripheral blood, bone marrow and spleen were harvested from transplanted mice. The tissues were passed through 100 um filters to achieve a single-cell suspension and red blood cells were lysed with ammonium chloride (RBC lysis buffer). Non-specific antibody staining was blocked with Trustain FX (BioLegend, #422302) for 10 minutes at room temperature. For primary engraftment studies cells were stained with the following antibodies: mTer119—PE-Cy5 (TER-119, eBiosciences, #15-5921-83); mCD45—PE-Cy7 (A20, eBioScience, #25-0453-82); hCD45—PacificBlue (Biolegend, #368539): hCD19—APC (HIB19, BD Biosciences, #555415); hCD33—PE (WM53, BD Biosciences, #555450); hCD14—BV711(M5E2, Biolegend, #301837). Dead cells were detected using Blue Reactive Dye (ThermoFisher #L34961) and excluded from analysis. For secondary engraftment studies, isolated bone marrow cells were stained with the following antibodies: mTer119—PE-Cy5 (TER-119, eBiosciences, #15-5921-83); mCD45—PE-Cy7 (A20, eBioScience, #25-0453-82): hCD45—PacificBlue (Biolegend, #368539); HLA-ABC-APC-Cy7 (W6/32. Biolegend, #311426); hCD19—APC (HIB19, BD Biosciences, #555415); hCD33—PE (WM53, BD Biosciences. #555450). Dead cells were detected using Propidium Iodine and excluded from study. Analysis was performed by flow cytometry on a BD FACSAria. Human engraftment was defined as the percentage of hCD45 among all (mouse or human) CD45⁺ cells.

Glucocerebrosidase Activity Assay

Glucocerebrosidase activity was assayed as previously described (83b). 100,000 to 200,00 cells were FAC-sorted to ensure same number of cells were being quantified. Protein was extracted by lysing cells in 200 μl of deionized water with a Branson Sonicator with probe, centrifuging lysates at 17,000×g for 10 minutes at 4° C., and collecting the supernatant containing the soluble proteins. Protein concentration in the supernatants was measured by Bradford assay kit with BSA standard curve ranging from 0.25-0.5 mg/ml (Thermo Scientific). To prepare the GCase assay working reagent, the fluorogenic substrate 4-methylumbeliferyl-β-d-glucopyranoside (Sigma, #M3633) was dissolved to a final concentration of 5 mM in citrate/phosphate buffer (pH 5.5) supplemented with 15% (w/v) sodium taurocholate. To perform the GCase assay, 25-50 μg protein extract (50 μL) was mixed with 100 μL of working reagent and incubated for 1 hour at 37° C. covered from light. Reactions were stopped with 200 μL stop buffer (0.2 M glycine/carbonate, pH 10.7). Fluorescence of 4-methylumbeliferone (4MU) liberated by GCase enzyme cleavage was measured using a Molecular Devices SpectraMax M3 multi-mode microplate reader with SoftMax Pro 5 software at excitation and emission wavelengths of 355 nm and 460 nm, respectively (top read). A standard curve for 4MU was established using 4MU sodium salt (Sigma) in assay buffer.

Immunocytochemistry and Imaging

Cells were seeded on coverslips 24-48 hours prior to analysis. All washes were performed with D-PBS (+calcium, +magnesium). Cells on coverslips were washed, fixed with 4% PFA in PBS for 30 min, permeabilized with 0.1% Triton-X in PBS for 10 minutes and blocked in 10% normal goat serum (NGS; Gibco) containing 0.25% Triton X-100 for 30 minutes at 25° C. After washing, coverslips were incubated in primary antibodies (α-CD68 (Abcam, ab31630) and α-Citrine (Abcam, ab19370) for 3-4 hours at 4° C. Primary antibodies were thoroughly washed and coverslips were incubated with secondary antibodies (FITC-α-mouse, APC-α-goat) for 1 hour covered from light. Coverslips were washed once more and mounted on glass coverslips with mounting media containing Hoechst die. Cells were imaged on a BZ-X710 Keyence fluorescence microscope.

Mice

NOD.Cg-PrkdcscidIL2rgtmlWjl/Sz (NSG) mice were developed at The Jackson Laboratory. NOD.Cg-Prkdcscid Il2rgtmlWjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ were described in Wunderlich et al., and Billerbeck et al. (45b,46b) and obtained from The Jackson Laboratory. Mice were housed in a 12-h dark/light cycle, temperature- and humidity-controlled environment with pressurized individually ventilated caging, sterile bedding, and unlimited access to sterile food and water in the animal barrier facility at Stanford University. All experiments were performed in accordance with National Institutes of Health institutional guidelines and were approved by the University Administrative Panel on Laboratory Animal Care (IACUC 20565 and 33365).

Tissue Macrophage Isolation

Peritoneal macrophages were isolated as single-cell suspension by injection of 6 mL of ice-cold PBS 1× in the peritoneal cavity, followed by aspiration of 4 mL of the peritoneal fluid, using syringe and 21 G needle. Liver and lung were dissected from mice after perfusion, minced and digested with 500 μg/mL Liberase™ (Roche, #05401119001) and 400 μg/mL DNase in RPMI media for 30 min at 37° C. After incubation, tissues were passed through 100 μm filters and washed twice. Liver samples were further processed by centrifugation in 33% Percoll Plus (GE Healthcare) for 15 min at 700 g, with brakes off. Red blood cells were lysed from cell pellets and a single cell suspension was prepared. For flow cytometry, non-specific antibody binding was blocked with TruStain FcX (Biolegend, #422302) and Cd16/cd32 anti-mouse (2.4G2, BD Biosciences, #553142). Cells were stained with hCD45-PacificBlue (Biolegend, #368539), mCD45—PE-Cy7 (A20, eBioScience, #25-0453-82), mTer119—PE-Cy5 (TER-119, eBiosciences, #15-5921-83) and h/mCD11b-PE (M1/70, BioLegend #101208). Dead cells were detected with Blue Reactive Dye (ThermoFisher #L34961).

Statistical Analysis

All statistical test including paired and unpaired t-tests, and one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test was performed using GraphPad Prism version 7 for Mac OS X, GraphPad Software, La Jolla Calif. USA. Data was reported as means when all conditions passed three normality tests (D'Agostino & Pearson, Shapiro-Wilk, and KS normality test).

REFERENCES

-   1b. Stimemann, J. et al. A Review of Gaucher Disease     Pathophysiology, Clinical Presentation and Treatments. International     journal of molecular sciences 18, doi:10.3390/ijms18020441 (2017). -   2b. Pastores, G. M. & Hughes, D. A. in GeneReviews® (eds M. P. Adam     et al.) University of Washington, Seattle. GeneReviews is a     registered trademark of the University of Washington, Seattle. All     rights reserved., 1993). -   3b. Charrow, J. et al. The Gaucher registry: demographics and     disease characteristics of 1698 patients with Gaucher disease. Arch     Intern Med 160, 2835-2843 (2000). -   4b. Sidransky, E. et al. Multicenter analysis of glucocerebrosidase     mutations in Parkinson's disease. N Engl J Med 361, 1651-1661,     doi:10.1056/NEJMoa0901281 (2009). -   5b. Ferraz, M. J. et al. Gaucher disease and Fabry disease: new     markers and insights in pathophysiology for two distinct     glycosphingolipidoses. Biochim Biophys Acta 1841, 811-825,     doi:10.1016/j.bbalip.2013.11.004 (2014). -   6b. Pandey. M. K. et al. Complement drives glucosylceramide     accumulation and tissue inflammation in Gaucher disease. Nature 543,     108-112, doi:10.1038/nature21368 (2017). -   7b. Pandey, M. K. et al. Gaucher disease: chemotactic factors and     immunological cell invasion in a mouse model. Molecular genetics and     metabolism 111, 163-171, doi:10.1016/j.ymgme.2013.09.002 (2014). -   8b. Deegan, P. et al. Treatment patterns from 647 patients with     Gaucher disease: An analysis from the Gaucher Outcome Survey. Blood     Cells Mol Dis 68, 218-225, doi:10.1016/j.bcmd.2016.10.014 (2018). -   9b. Hughes, D. A. et al. Velaglucerase alfa (VPRIV) enzyme     replacement therapy in patients with Gaucher disease: Long-term data     from phase III clinical trials. American journal of hematology 90,     584-591, doi:10.1002/ajh.24012 (2015). -   10b. Lukina, E. et al. Improvement in hematological, visceral, and     skeletal manifestations of Gaucher disease type 1 with oral     eliglustat tartrate (Genz-112638) treatment: 2-year results of a     phase 2 study. Blood 116, 4095-4098,     doi:10.1182/blood-2010-06-293902 (2010). -   11b. Pastores, G. M., Barnett, N. L. & Kolodny, E. H. An open-label,     noncomparative study of miglustat in type I Gaucher disease:     efficacy and tolerability over 24 months of treatment. Clin Ther 27,     1215-1227. doi:10.1016/j.clinthera.2005.08.004 (2005). -   12b. Weinreb, N. J. et al. Long-term clinical outcomes in type 1     Gaucher disease following 10 years of imiglucerase treatment. J     Inherit Metab Dis 36, 543-553, doi:10.1007/s10545-012-9528-4 (2013). -   13b. Zimran, A., Wajnrajch, M., Hemandez, B. & Pastores, G. M.     Taliglucerase alfa: safety and efficacy across 6 clinical studies in     adults and children with Gaucher disease. Orphanet journal of rare     diseases 13, 36, doi:10.1186/s13023-018-0776-8 (2018). -   14b. Ito, S. & Barrett, A. J. Gauchers disease—a reappraisal of     hematopoietic stem cell transplantation. Pediatric hematology and     oncology 30, 61-70, doi:10.3109/08880018.2012.762076 (2013). -   15b. Machaczka, M. Allogeneic hematopoietic stem cell     transplantation for treatment of Gaucher disease. Pediatric     hematology and oncology 30, 459-461,     doi:10.3109/08880018.2013.793757 (2013). -   16b. Somaraju, U. R. & Tadepalli, K. Hematopoietic stem cell     transplantation for Gaucher disease. The Cochrane database of     systematic reviews 10. Cd006974, doi:10.1002/14651858.CD006974.pub4     (2017). -   17b. Correll, P. H., Colilla, S., Dave, H. P. & Karlsson, S. High     levels of human glucocerebrosidase activity in macrophages of     long-term reconstituted mice after retroviral infection of     hematopoietic stem cells. Blood 80, 331-336 (1992). -   18b. Dunbar, C. E. et al. Retroviral transfer of the     glucocerebrosidase gene into CD34+ cells from patients with Gaucher     disease: in vivo detection of transduced cells without     myeloablation. Human gene therapy 9, 2629-2640,     doi:10.1089/hum.1998.9.17-2629 (1998). -   19b. Krall, W. J., Challita, P. M., Perlmutter, L. S.,     Skelton, D. C. & Kohn, D. B. Cells expressing human     glucocerebrosidase from a retroviral vector repopulate macrophages     and central nervous system microglia after murine bone marrow     transplantation. Blood 83, 2737-2748 (1994). -   20b. Schiffmann, R. et al. Transfer of the human glucocerebrosidase     gene into hematopoietic stem cells of nonablated recipients:     successful engraftment and long-term expression of the transgene.     Blood 86, 1218-1227 (1995). -   21b. Kim, E. Y. et al. Long-term expression of the human     glucocerebrosidase gene in vivo after transplantation of     bone-marrow-derived cells transformed with a lentivirus vector. The     journal of gene medicine 7, 878-887, doi:10.1002/jgm.732 (2005). -   22b. Enquist, I. B. et al. Effective cell and gene therapy in a     murine model of Gaucher disease. Proceedings of the National Academy     of Sciences of the United States of America 103, 13819-13824,     doi:10.1073/pnas.0606016103 (2006). -   23b. Dahl, M. et al. Lentiviral gene therapy using cellular     promoters cures type 1 Gaucher disease in mice. Mol Ther 23,     835-844, doi:10.1038/mt.2015.16 (2015). -   24b. McCormack, M. P. & Rabbitts, T. H. Activation of the T-cell     oncogene LMO2 after gene therapy for X-linked severe combined     immunodeficiency. N Engl J Med 350, 913-922,     doi:10.1056/NEJMra032207 (2004). -   25b. Ranzani, M. et al. Lentiviral vector-based insertional     mutagenesis identifies genes associated with liver cancer. Nat     Methods 10, 155-161, doi:10.1038/nmeth.2331 (2013). -   26b. Porteus, M. H. A New Class of Medicines through DNA Editing. N     Engl J Med 380, 947-959, doi:10.1056/NEJMral800729 (2019). -   27b. Bak, R. O., Dever. D. P. & Porteus. M. H. CRISPR/Cas9 genome     editing in human hematopoietic stem cells. Nat Protoc 13, 358-376,     doi:10.1038/nprot.2017.143 (2018). -   28b. Dever, D. P. et al. CRISPR/Cas9 beta-globin gene targeting in     human haematopoietic stem cells. Nature 539, 384-389,     doi:10.1038/nature20134 (2016). -   29b. Gomez-Ospina, N. et al. Human genome-edited hematopoietic stem     cells phenotypically correct Mucopolysaccharidosis type I. Nature     Communications, doi:10.1038/s41467-019-11962-8 (2019). -   30b. Pavel-Dinu, M. et al. Gene correction for SCID-X1 in long-term     hematopoietic stem cells. Nat Commun 10, 1634,     doi:10.1038/s41467-019-09614-y (2019). -   31b. Hendel, A. et al. Chemically modified guide RNAs enhance     CRISPR-Cas genome editing in human primary cells. Nat Biotechnol 33,     985-989, doi:10.1038/nbt.3290 (2015). -   32b. Bak, R. O., Gomez-Ospina, N. & Porteus, M. H. Gene Editing on     Center Stage. Trends Genet 34, 600-611,     doi:10.1016/j.tig.2018.05.004 (2018). -   33b. Lombardo, A. et al. Site-specific integration and tailoring of     cassette design for sustainable gene transfer. Nat Methods 8,     861-869, doi:10.1038/nmeth.1674 (2011). -   34b. Sadelain, M., Papapetrou, E. P. & Bushman, F. D. Safe harbours     for the integration of new DNA in the human genome. Nature reviews.     Cancer 12, 51-58. doi:10.1038/nrc3179 (2012). -   35b. Novembre, J., Galvani, A. P. & Slatkin, M. The geographic     spread of the CCR5 Delta32 HIV-resistance allele. PLoS Biol 3, e339,     doi:10.1371/journal.pbio.0030339 (2005). -   36b. Samson, M. et al. Resistance to HIV-1 infection in caucasian     individuals bearing mutant alleles of the CCR-5 chemokine receptor     gene. Nature 382, 722-725, doi:10.1038/382722a0 (1996). -   37b. Bak, R. O. et al. Multiplexed genetic engineering of human     hematopoietic stem and progenitor cells using CRISPR/Cas9 and AAV6.     Elife 6, doi:10.7554/eLife.27873 (2017). -   38b. Gough, P. J., Gordon, S. & Greaves, D. R. The use of human CD68     transcriptional regulatory sequences to direct high-level expression     of class A scavenger receptor in macrophages in vitro and in vim.     Immunology 103, 351-361, doi:10.1046/j.1365-2567.2001.01256.x     (2001). -   39b. Gough, P. J. & Raines, E. W. Gene therapy of apolipoprotein     E-deficient mice using a novel macrophage-specific retroviral     vector. Blood 101, 485-491, doi:10.1182/blood-2002-07-2131 (2003). -   40b. Levin, M. C. et al. Evaluation of macrophage-specific promoters     using lentiviral delivery in mice. Gene Ther 19, 1041-1047,     doi:10.1038/gt.2011.195 (2012). -   41b. Way, K. J. et al. The generation and properties of human     macrophage populations from hemopoietic stem cells. J Leukoc Biol     85, 766-778, doi: 10.1189/jlb.1108689 (2009). -   42b. McDermott, S. P., Eppert, K., Lechman, E. R., Doedens, M. &     Dick, J. E. Comparison of human cord blood engraftment between     immunocompromised mouse strains. Blood 116, 193-200.     doi:10.1182/blood-2010-02-271841 (2010). -   43b. Wiekmeijer, A. S. et al. Sustained Engraftment of Cryopreserved     Human Bone Marrow CD34(+) Cells in Young Adult NSG Mice. Biores Open     Access 3, 110-116, doi:10.1089/biores.2014.0008 (2014). -   44b. Manz, M. G. Human-hemato-lymphoid-system mice: opportunities     and challenges. Immunity 26, 537-541,     doi:10.1016/j.immuni.2007.05.001 (2007). -   45b. Billerbeck, E. et al. Development of human CD4+FoxP3+     regulatory T cells in human stem cell factor-,     granulocyte-macrophage colony-stimulating factor-, and     interleukin-3-expressing NOD-SCID IL2Rgamma(null) humanized mice.     Blood 117, 3076-3086, doi:10.1182/blood-2010-08-301507 (2011). -   46b. Wunderlich, M. et al. AML xenograft efficiency is significantly     improved in NOD/SCID-IL2RG mice constitutively expressing human SCF,     GM-CSF and IL-3. Leukemia 24, 1785-1788, doi:10.1038/leu.2010.158     (2010). -   47b. Charrow, J., Dulisse, B., Grabowski, G. A. & Weinreb, N. J. The     effect of enzyme replacement therapy on bone crisis and bone pain in     patients with type 1 Gaucher disease. Clinical genetics 71, 205-211,     doi:10.1111/j.1399-0004.2007.00769.x (2007). -   48b. Cohen, I. J. et al. Low-dose high-frequency enzyme replacement     therapy prevents fractures without complete suppression of painful     bone crises in patients with severe juvenile onset type I Gaucher     disease. Blood Cells Mol Dis 24, 296-302, doi:10.1006/bcmd.1998.0195     (1998). -   49b. Deegan, P. B. et al. Osseous manifestations of adult Gaucher     disease in the era of enzyme replacement therapy. Medicine 90,     52-60, doi:10.1097/MD.0b013e3182057be4 (2011). -   50b. Ryan, E., Seehra, G., Sharma, P. & Sidransky, E.     GBA1-associated parkinsonism: new insights and therapeutic     opportunities. Current opinion in neurology,     doi:10.1097/wco.0000000000000715 (2019). -   51b. Wilke, M. et al. Evaluation of the frequency of non-motor     symptoms of Parkinson's disease in adult patients with Gaucher     disease type 1. Orphanet journal of rare diseases 14, 103,     doi:10.1186/s13023-019-1079-4 (2019). -   52b. Brady, R. O. et al. Management of neutralizing antibody to     Ceredase in a patient with type 3 Gaucher disease. Pediatrics 100,     E11, doi:10.1542/peds.100.6.e11 (1997). -   53b. Germain, D. P., Kaneski, C. R. & Brady, R. O. Mutation analysis     of the acid beta-glucosidase gene in a patient with type 3 Gaucher     disease and neutralizing antibody to alglucerase. Mutation research     483, 89-94 (2001). -   54b. Ponce, E., Moskovitz, J. & Grabowski, G. Enzyme therapy in     Gaucher disease type 1: effect of neutralizing antibodies to acid     beta-glucosidase. Blood 90, 43-48 (1997). -   55b. Starzyk, K., Richards. S., Yee, J., Smith, S. E. & Kingma. W.     The long-term international safety experience of imiglucerase     therapy for Gaucher disease. Molecular genetics and metabolism 90,     157-163, doi:10.1016/j.ymgme.2006.09.003 (2007). -   56b. Zhao, H., Bailey, L. A. & Grabowski, G. A. Enzyme therapy of     gaucher disease: clinical and biochemical changes during production     of and tolerization for neutralizing antibodies. Blood Cells Mol Dis     30, 90-96 (2003). -   57b. Cox, T. M. et al. Evaluation of miglustat as maintenance     therapy after enzyme therapy in adults with stable type 1 Gaucher     disease: a prospective, open-label non-inferiority study. Orphanet     journal of rare diseases 7, 102, doi:10.1186/1750-1172-7-102 (2012). -   58b. Beurtler, E. The treatment of Gaucher disease in countries with     limited health care resources. Indian Joumal of Human Genetics 11,     121-127 (2005). -   59b. Nalysnyk, L., Sugarman, R., Cele, C., Uyei, J. & Ward, A.     Budget Impact Analysis of Eliglustat for the Treatment of Gaucher     Disease Type 1 in the United States. Journal of managed care &     specialty pharmacy 24, 1002-1008, doi:10.18553/jmcp.2018.24.10.1002     (2018). -   60b. van Dussen, L., Biegstraaten, M., Hollak, C. E. &     Dijkgraaf, M. G. Cost-effectiveness of enzyme replacement therapy     for type 1 Gaucher disease. Orphanet journal of rare diseases 9, 51,     doi:10.1186/1750-1172-9-51 (2014). -   61b. Gassas, A. et al. Long-term adaptive functioning outcomes of     children with inherited metabolic and genetic diseases treated with     hematopoietic stem cell transplantation in a single large pediatric     center: parents' perspective. Journal of pediatric     hematology/oncology 33, 216-220. doi:10.1097/MPH.0b013e3182050945     (2011). -   62b. Hoogerbrugge, P. M. et al. Allogeneic bone marrow     transplantation for lysosomal storage diseases. The European Group     for Bone Marrow Transplantation. Lancet (London, England) 345,     1398-1402, doi:10.1016/s0140-6736(95)92597-x (1995). -   63b. Starer. F., Sargent, J. D. & Hobbs, J. R. Regression of the     radiological changes of Gaucher's disease following bone marrow     transplantation. The British journal of radiology 60, 1189-1195,     doi:10.1259/0007-1285-60-720-1189 (1987). -   64b. Yabe, H. et al. Secondary G-CSF mobilized blood stem cell     transplantation without preconditioning in a patient with Gaucher     disease; Report of a new approach which resulted in complete     reversal of severe skeletal involvement. The Tokai journal of     experimental and clinical medicine 30, 77-82 (2005). -   65b. Yen, C. C., Chiou, T. J., Lin, C. Y., Wang, N. H. & Chen, P. M.     Allogeneic bone marrow transplantation for Gaucher disease—a case     report. Zhonghua yi xue za zhi=Chinese medical journal; Free China     ed 59, 372-376 (1997). -   66b. Erikson, A. et al. Clinical and biochemical outcome of marrow     transplantation for Gaucher disease of the Norrbottnian type. Acta     paediatrica Scandinavica 79, 680-685 (1990). -   67b. Rappeport, J. M. & Ginns, E. I. Bone-marrow transplantation in     severe Gaucher's disease. N Engl J Med 311, 84-88.     doi:10.1056/nejm198407123110203 (1984). -   68b. Svennerholm, L., Erikson, A., Groth, C. G., Ringden, O. &     Mansson, J. E. Norrbottnian type of Gaucher disease—clinical,     biochemical and molecular biology aspects: successful treatment with     bone marrow transplantation. Developmental neuroscience 13, 345-351,     doi:10.1159/000112184 (1991). -   69b. Tsai, P. et al. Allogenic bone marrow transplantation in severe     Gaucher disease. Pediatric research 31, 503-507,     doi:10.1203/00006450-199205000-00019 (1992). -   70b. Sharma. R. et al. In vivo genome editing of the albumin locus     as a platform for protein replacement therapy. Blood 126, 1777-1784,     doi:10.1182/blood-2014-12-615492 (2015). -   71b. Galvani, A. P. & Slatkin, M. Evaluating plague and smallpox as     historical selective pressures for the CCR5-Delta 32 HIV-resistance     allele. Proceedings of the National Academy of Sciences of the     United States of America 100, 15276-15279,     doi:10.1073/pnas.2435085100 (2003). -   72b. Falcon, A. et al. CCR5 deficiency predisposes to fatal outcome     in influenza virus infection. The Journal of general virology 96,     2074-2078, doi:10.1099/vir.0.000165 (2015). -   73b. Cahill, M. E., Conley, S., DeWan, A. T. & Montgomery, R. R.     Identification of genetic variants associated with dengue or West     Nile virus disease: a systematic review and meta-analysis. BMC     infectious diseases 18, 282, doi:10.1186/s12879-018-3186-6 (2018). -   74b. Horowitz, M. et al. The human glucocerebrosidase gene and     pseudogene: structure and evolution. Genomics 4, 87-96 (1989). -   75b. Visigalli, I. et al. The galactocerebrosidase enzyme     contributes to the maintenance of a functional hematopoietic stem     cell niche. Blood 116, 1857-1866, doi:10.1182/blood-2009-12-256461     (2010). -   76b. Naldini, L. Gene therapy returns to centre stage. Nature 526,     351-360, doi:10.1038/nature15818 (2015). -   77b. Mazurier, F., Gan. O. I., McKenzie, J. L., Doedens, M. &     Dick, J. E. Lentivector-mediated clonal tracking reveals intrinsic     heterogeneity in the human hematopoietic stem cell compartment and     culture-induced stem cell impairment. Blood 103, 545-552,     doi:10.1182/blood-2003-05-1558 (2004). -   78b. Chan, K. W., Wong. L. T., Applegarth, D. & Davidson. A. G. Bone     marrow transplantation in Gaucher's disease: effect of mixed     chimeric state. Bone marrow transplantation 14, 327-330 (1994). -   79b. Ringden, O. et al. Ten years' experience of bone marrow     transplantation for Gaucher disease. Transplantation 59, 864-870     (1995). -   80b. Enquist, I. B. et al. Successful low-risk hematopoietic cell     therapy in a mouse model of type I Gaucher disease. Stem cells     (Dayton, Ohio) 27, 744-752, doi:10.1634/stemcells.2008-0844 (2009). -   81b. Dahl, M. et al. Lentiviral gene therapy using cellular     promoters cures type 1 Gaucher disease in mice. Mol Ther 23,     835-844, doi:10.1038/mt.2015.16 (2015). -   82b. Khan, I. F., Hirata, R. K. & Russell, D. W. AAV-mediated gene     targeting methods for human cells. Nature protocols 6, 482-501,     doi:10.1038/nprot.2011.301 (2011). -   83b. Daniels, L. B., Glew, R H., Diven, W. F., Lee, R. E. &     Radin, N. S. An improved fluorometric leukocyte beta-glucosidase     assay for Gaucher's disease. Clin Chim Acta 115, 369-375,     doi:10.1016/0009-8981(81)90251-5 (1981).

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference.

Informal Sequence Listing SEQ ID NO. 1 Left homology arm of CCR5 constructs: chr3:46,372,658-46,373,157. Assembly hg38 TTTCATGAATTCCCCCAACAGAGCCAAGCTCTCCATCTAGTGGACAGGGAAGCTA GCAGCAAACCTTCCCTTCACTACAAAACTTCATTGCTTGGCCAAAAAGAGAGTTA ATTCAATGTAGACATCTATGTAGGCAATTAAAAACCTATTGATGTATAAAACAGT TTGCATTCATGGAGGGCAACTAAATACATTCTAGGACTTTATAAAAGATCACTTT TTATTTATGCACAGGGTGGAACAAGATGGATTATCAAGTGTCAAGTCCAATCTAT GACATCAATTATTATACATCGGAGCCCTGCCAAAAAATCAATGTGAAGCAAATC GCAGCCCGCCTCCTGCCTCCGCTCTACTCACTGGTGTTCATCTTTGGTTTTGTGGG CAACATGCTGGTCATCCTCATCCTGATAAACTGCAAAAGGCTGAAGAGCATGACT GACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTTCCTTCTTACTGTCCC CTTC SEQ ID NO. 2 Right homology arm of CCR5 constructs: chr3:46,373,158-46,373,657. Assembly hg38 TGGGCTCACTATGCTGCCGCCCAGTGGGACTTTGGAAATACAATGTGTCAACTCT TGACAGGGCTCTATTTTATAGGCTTCTTCTCTGGAATCTTCTTCATCATCCTCCTG ACAATCGATAGGTACCTGGCTGTCGTCCATGCTGTGTTTGCTTTAAAAGCCAGGA CGGTCACCTTTGGGGTGGTGACAAGTGTGATCACTTGGGTGGTGGCTGTGTTTGC GTCTCTCCCAGGAATCATCTTTACCAGATCTCAAAAAGAAGGTCTTCATTACACC TGCAGCTCTCATTTTCCATACAGTCAGTATCAATTCTGGAAGAATTTCCAGACATT AAAGATAGTCATCTTGGGGCTGGTCCTGCCGCTGCTTGTCATGGTCATCTGCTAC TCGGGAATCCTAAAAACTCTGCTTCGGTGTCGAAATGAGAAGAAGAGGCACAGG GCTGTGAGGCTTATCTTCACCATCATGATTGTTTATTTTCTCTTCTGGGCTCCCTA CAA SEQ ID NO: 3 CCR5 sgRNA target sequence (with PAM sequence) GCAGCATAGTGAGCCCAGAAGGG SEQ ID NO: 4 CCR5 sgRNA target sequence (without PAM sequence) GCAGCATAGTGAGCCCAGAA SEQ ID NO: 5 sgRNA sequence 5′-2′OMe(G(ps)C(ps)A(ps))GCA UAG UGA GCC CAG AAG UUU UAG AGC UAG AAA UAG CAA GUU AAA AUA AGG CUA GUC CGU UAU CAA CUU GAA AAA GUG GCA CCG AGU CGG UGC UU 2′OMe(U(ps)U(ps)U(ps))U-3′ (ps) indicates phosphorothioate SEQ ID NO: 6 MPS1: Strategy is to knock in IDUA cDNA into Exon 3 of CCR5 safe harbor gene to overexpress IDUA enzyme and the selectable marker tNGFR. Otherwise identical or substantially identical sequences in which the INGFR marker (nucleotides 2961-3848) is replaced with another selectable can also be used. Left homology arm: 1-500 bp PGK promoter: 501-1001 bp IDUA CDNA: 1002-2960 bp T2A-tNGFR: 2961-3848 bp BgH Poly A: 3849-4099 bp Right homology arm: 4100-4599 bp TTTCATGAATTCCCCCAACAGAGCCAAGCTCTCCATCTAGTGGACAGGGAAGCTA GCAGCAAACCTTCCCTTCACTACAAAACTTCATTGCTTGGCCAAAAAGAGAGTTA ATTCAATGTAGACATCTATGTAGGCAATTAAAAACCTATTGATGTATAAAACAGT TTGCATTCATGGAGGGCAACTAAATACATTCTAGGACTTTATAAAAGATCACTTT TTATTTATGCACAGGGTGGAACAAGATGGATTATCAAGTTGTCAAGTCCAATCTAT GACATCAATTATTATACATCGGAGCCCTGCCAAAAAATCAATGTGAAGCAAATC GCAGCCCGCCTCCTGCCTCCGCTCTACTCACTGGTGTTCATCTTTGGTTTTGTGGG CAACATGCTGGTCATCCTCATCCTGATAAACTGCAAAAGGCTGAAGAGCATGACT GACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTTCCTTCTTACTGTCCC CTTCTctagataccgggtaggggaggcgcttttcccaaggcagtctggagcatgcgctttagcagccccgetgggcacttggcgc tacacaagtggcctctggcctcgcacacattccacatccaccggtaggcgccaaccggctccgttctttggtggccccttcgcgccac cttctactcctcccctagtcaggaagttcccccccgccccgcagctcgcgtcgtgcaggacgtgacaaatggaagtagcacgtctcac tagtctcgtgcagatggacagcaccgctgagcaatggaagcgggtaggcctttggggcagcggccaatagcagctttgctccttcgct ttctgggctcagaggctgggaaggggtgggtccggggggggctcagggggggctcaggggggggcgggcgcccgaaggt cctccggaggcccggcattctgcacgcttcaaaagcgcacgtctgccgcgctgttctcctcttcctcaggatccATGCGTCCC CTGCGCCCCCGCGCCGCGCTGCTGGCGCTCCTGGCCTCGCTCCTGGCCGCGCCCC CGGTGGCCCCGGCCGAGGCCCCGCACCTGGTGCATGTGGACGCGGCCCGCGCGC TGTGGCCCCTGCGGCGCTTCTGGAGGAGCACAGGCTTCTGCCCCCCGCTGCCACA CAGCCAGGCTGACCAGTACGTCCTCAGCTGGGACCAGCAGCTCAACCTCGCCTAT GTGGGCGCCGTCCCTCACCGCGGCATCAAGCAGGTCCGGACCCACTGGCTGCTG GAGCTTGTCACCACCAGGGGGTCCACTGGACGGGGCCTGAGCTACAACTTCACC CACCTGGACGGGTACCTGGACCTTCTCAGGGAGAACCAGCTCCTCCCAGGGTTTG AGCTGATGGGCAGCGCCTCGGGCCACTTCACTGACTTTGAGGACAAGCAGCAGG TGTTTGAGTGGAAGGACTTGGTCTCCAGCCTGGCCAGGAGATACATCGGTAGGTA CGGACTGGCGCATGTTTCCAAGTGGAACTTCGAGACGTGGAATGAGCCAGACCA CCACGACTTTGACAACGTCTCCATGACCATGCAAGGCTTCCTGAACTACTACGAT GCCTGCTCGGAGGGTCTGCGCGCCGCCAGCCCCGCCCTGCGGCTGGGAGGCCCC GGCGACTCCTTCCACACCCCACCGCGATCCCCGCTGAGCTGGGGCCTCCTGCGCC ACTGCCACGACGGTACCAACTTCTTCACTGGGGAGGCGGGCGTGCGGCTGGACT ACATCTCCCTCCACAGGAAGGGTGCGCGCAGCTCCATCTCCATCCTGGAGCAGGA GAAGGTCGTCGCGCAGCAGATCCGGCAGCTCTTCCCCAAGTTCGCGGACACCCCC ATTTACAACGACGAGGCGGACCCGCTGGTGGGCTGGTCCCTGCCACAGCCGTGG AGGGCGGACGTGACCTACGCGGCCATGGTGGTGAAGGTCATCGCGCAGCATCAG AACCTGCTACTGGCCAACACCACCTCCGCCTTCCCCTACGCGCTCCTGAGCAACG ACAATGCCTTCCTGAGCTACCACCCGCACCCCTTCGOGCAGCGCACGCTCACCGC GCGCTTCCAGGTCAACAACACCCGCCCGCCGCACGTGCAGCTGTTGCGCAAGCC GGTGCTCACGGCCATGGGGCTGCTGGCGCTGCTGGATGAGGAGCAGCTCTGGGC CGAAGTGTCGCAGGCCGGGACCGTCCTGGACAGCAACCACACGGTGGGCGTCCT GGCCAGCGCCCACCGCCCCCAGGGCCCGGCCGACGCCTGGCGCGCCGCGGTGCT GATCTACGCGAGCGACGACACCCGCGCCCACCCCAACCGCAGCGTCGCGGTGAC CCTGCGGCTGCGCGGGGTGCCCCCCGGCCCGGGCCTGGTCTACGTCACGCGCTAC CTGGACAACGGGCTCTGCAGCCCCGACGGCGAGTGGCGGCGCCTGGGCCGGCCC GTCTTCCCCACGGCAGAGCAGTTCCGGCGCATGCGCGCGGCTGAGGACCCGGTG GCCGCGGCGCCCCGCCCCTTACCCGCCGGCGGCCGCCTGACCCTCAGACCTGCAC TTAGATTGCCTTCCCTTTTGTTGGTCCACGTTTGCGCTAGGCCCGAGAAACCGCCA GGACAAGTAACACGGCTTCGGGCGCTGCCACTTACTCAGGGGCAGCTGGTGCTG GTTTGGTCAGACGAGCATGTCGGAAGCAAATGCCTTTGGACCTACGAGATACAA TTTTCACAGGATGGTAAGGCTTACACTCCGGTCTCAAGAAAGCCCAGTACCTTTA ACCTTTTTGTGTTCAGTCCAGATACTGGAGCAGTAAGCGGTTCATATAGAGTCAG AGCGCTGGATTACTGGGCCAGGCCCGGACCTTTCTCAGATCCGGTCCCCTACCTG GAAGTTCCCGTGCCGCGGGGTCCTCCATCACCAGGCAACCCAGGAAGCGGAGCT ACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCT GGGGCAGGTGCCACCGGCCGCGCCATGGACGGGCCGCGCCTGCTGCTGTTGCTG CTTCTGGGGGTGTCCCTTGGAGGTGCCAAGGAGGCATGCCCCACAGGCCTGTACA CACACAGCGGTGAGTGCTGCAAAGCCTGCAACCTGGGCGAGGGTGTGGCCCAGC CTTGTGGAGCCAACCAGACCGTGTGTGAGCCCTGCCTGGACAGCGTGACGTTCTC CGACGTGGTGAGCGCGACCGAGCCGTGCAAGCCGTGCACCGAGTGCGTGGGGCT CCAGAGCATGTCGGCGCCaTGCGTGGAGGCCGACGACGCCGTGTGCCGCTGCGCC TACGGCTACTACCAGGATGAGACGACTGGGCGCTGCGAGGCGTGCCGCGTGTGC GAGGCGGGCTCGGGCCTCGTGTTCTCCTGCCAGGACAAGCAGAACACCGTGTGC GAGGAGTGCCCCGACGGCACGTATTCCGACGAGGCCAACCACGTGGACCCGTGC CTGCCCTGCACCGTGTGCGAGGACACCGAGCGCCAGCTCCGCGAGTGCACACGC TGGGCCGACGCCGAGTGCGAGGAGATCCCTGGCCGTTGGATTACACGGTCCACA CCCCCAGAGGGCTCGGACAGCACAGCCCCCAGCACCCAGGAGCCTGAGGCACCT CCAGAACAAGACCTCATAGCCAGCACGGTGGCGGGTGTGGTGACCACAGTGATG GGCAGCTCCCAGCCCGTGGTGACCCGAGGCACCACCGACAACCTCATCCCTGTCT ATTGCTCCATCCTGGCTGCTGTGGTTGTGGGTCTTGTGGCCTACATAGCCTTCAAG AGGTAAtaacTCGAGCCGCTGAtcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgc cttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattct ggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctactagtt gggctcactatgctgccgcccagtgggactttggaaatacaatgtgtcaactcttgacagggctctattttataggcttcttctctggaatct tcttcatcatcctcctgacaatcgataggtacctggctgtcgtccatgctgtgtttgctttaaaagccaggacggtcacctttggggtggtg acaagtgtgatcacttgggtggtggctgtgtttgcgtctctcccaggaatcatctttaccagatctcaaaaagaaggtcttcattacacctg cagctctcattttccatacagtcagtatcaattctggaagaatttccagacattaaagatagtcatcttggggctggtcctgccgctgcttgt catggtcatctgctactcgggaatcctaaaaactctgcttcggtgtcgaaatgagaagaagaggcacagggctgtgaggcttatcttca ccatcatgattgtttattttctcttctgggctccctacaa SEQ ID NO: 7 MPS1: Strategy is to knock in IDUA cDNA into Exon 3 of CCR5 safe harbor gene to overexpress IDUA enzyme without a selectable marker Left homology arm: 1-500 bp PGK promoter: 507-995 bp IDUA cDNA: 1002-2960 bp BgH Poly A: 2987-3194 bp Right homology arm: 3212-3711 bp TTTCATGAATTCCCCCAACAGAGCCAAGCTCTCCATCTAGTGGACAGGGAAGCTA GCAGCAAACCTTCCCTTCACTACAAAACTTCATTGCTTGGCCAAAAAGAGAGTTA ATTCAATGTAGACATCTATGTAGGCAATTAAAAACCTATTGATGTATAAAACAGT TTGCATTCATGGAGGGCAACTAAATACATTCTAGGACTTTATAAAAGATCACTTT TTATTTATGCACAGGGTGGAACAAGATGGATTATCAAGTGTCAAGTCCAATCTAT GACATCAATTATTATACATCGGAGCCCTGCCAAAAAATCAATGTGAAGCAAATC GCAGCCCGCCTCCTGCCTCCGCTCTACTCACTGGTGTTCATCTTTGGTTTTGTGGG CAACATGCTGGTCATCCTCATCCTGATAAACTGCAAAAGGCTGAAGAGCATGACT GACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTTCCTTCTTACTGTCCC CTTCTctagataccgggtaggggaggcgcttttcccaaggcagtctggagcatgcgctttagcagccccgctgggcacttggcgc tacacaagtggcctctggcctcgcacacattccacatccaccggtaggcgccaaccggctccgttctttggtggccccttcgcgccac cttctactcctcccctagtcaggaagttcccccccgccccgcagctcgcgtcgtgcaggacgtgacaaatggaagtagcacgtctcac tagtctcgtgcagatggacagcaccgctgagcaatggaagcgggtaggcctttggggcagcggccaatagcagctttgctccttcgct ttctgggctcagaggctgggaaggggtgggtccggggggggctcagggggggctcaggggggggcgggcgcccgaaggt CTGCGCCCCCGCGCCGCGCTGCTGGCGCTCCTGGCCTCGCTCCTGGCCGCGCCCC CGGTGGCCCCGGCCGAGGCCCCGCACCTGGTGCATGTGGACGCGGCCCGCGCGC TGTGGCCCCTGCGGCGCTTCTGGAGGAGCACAGGCTTCTGCCCCCCGCTGCCACA CAGCCAGGCTGACCAGTACGTCCTCAGCTGGGACCAGCAGCTCAACCTCGCCTAT GTGGGCGCCGTCCCTCACCGCGGCATCAAGCAGGTCCGGACCCACTGGCTGCTG GAGCTTGTCACCACCAGGGGGTCCACTGGACGGGGCCTGAGCTACAACTTCACC CACCTGGACGGGTACCTGGACCTTCTCAGGGAGAACCAGCTCCTCCCAGGGTTTG AGCTGATGGGCAGCGCCTCGGGCCACTTCACTGACTTTGAGGACAAGCAGCAGG TGTTTGAGTGGAAGGACTTGGTCTCCAGCCTGGCCAGGAGATACATCGGTAGGTA CGGACTGGCGCATGTTTCCAAGTGGAACTTCGAGACGTGGAATGAGCCAGACCA CCACGACTTTGACAACGTCTCCATGACCATGCAAGGCTTCCTGAACTACTACGAT GCCTGCTCGGAGGGTCTGCGCGCCGCCAGCCCCGCCCTGCGGCTGGGAGGCCCC GGCGACTCCTTCCACACCCCACCGCGATCCCCGCTGAGCTGGGGCCTCCTGCGCC ACTGCCACGACGGTACCAACTTCTTCACTGGGGAGGCGGGCGTGCGGCTGGACT ACATCTCCCTCCACAGGAAGGGTGCGCGCAGCTCCATCTCCATCCTGGAGCAGGA GAAGGTCGTCGCGCAGCAGATCCGGCAGCTCTTCCCCAAGTTCGCGGACACCCCC ATTTACAACGACGAGGCGGACCCGCTGGTGGGCTGGTCCCTGCCACAGCCGTGG AGGGCGGACGTGACCTACGCGGCCATGGTGGTGAAGGTCATCGCGCAGCATCAG AACCTGCTACTGGCCAACACCACCTCCGCCTTCCCCTACGCGCTCCTGAGCAACG ACAATGCCTTCCTGAGCTACCACCCGCACCCCTTCGCGCAGCGCACGCTCACCGC GCGCTTCCAGGTCAACAACACCCGCCCGCCGCACGTGCAGCTGTTGCGCAAGCC GGTGCTCACGGCCATGGGGCTGCTGGCGCTGCTGGATGAGGAGCAGCTCTGGGC CGAAGTGTCGCAGGCCGGGACCGTCCTGGACAGCAACCACACGGTGGGCGTCCT GGCCAGCGCCCACCGCCCCCAGGGCCCGGCCGACGCCTGGCGCGCCGCGGTGCT GATCTACGCGAGCGACGACACCCGCGCCCACCCCAACCGCAGCGTCGCGGTGAC CCTGCGGCTGCGCGGGGTGCCCCCCGGCCCGGGCCTGGTCTACGTCACGCGCTAC CTGGACAACGGGCTCTGCAGCCCCGACGGCGAGTGGCGGCGCCTGGGCCGGCCC GTCTTCCCCACGGCAGAGCAGTTCCGGCGCATGCGCGCGGCTGAGGACCCGGTG GCCGCGGCGCCCCGCCCCTTACCCGCCGGCGGCCGCCTGACCCTGCGCCCCGCGC TGCGGCTGCCGTCGCTTTTGCTGGTGCACGTGTGTGCGCGCCCCGAGAAGCCGCC CGGGCAGGTCACGCGGCTCCGCGCCCTGCCCCTGACCCAAGGGCAGCTGGTTCTG GTCTGGTCGGATGAACACGTGGGCTCCAAGTGCCTGTGGACATACGAGATCCAG TTCTCTCAGGACGGTAAGGCGTACACCCCGGTCAGCAGGAAGCCATCGACCTTCA ACCTCTTTGTGTTCAGCCCAGACACAGGTGCTGTCTCTGGCTCCTACCGAGTTCG AGCCCTGGACTACTGGGCCCGACCAGGCCCCTTCTCGGACCCTGTGCCGTACCTG GAGGTCCCTGTGCCAAGAGGGCCCCCATCCCCGGGCAATCCATAGcTCGAGCCGC TGAtcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactccc actgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagc aagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctactagttgggctcactatgctgccgcccagtggg actttggaaatacaatgtgtcaactcttgacagggctctattttataggcttcttctctggaatcttcttcatcatcctcctgacaatcgatagg tacctggctgtcgtccatgctgtgtttgctttaaaagccaggacggtcacctttggggtggtgacaagtgtgatcacttgggtggtggctg tgtttgcgtctctcccaggaatcatctttaccagatctcaaaaagaaggtcttcattacacctgcagctctcattttccatacagtcagtatca attctggaagaatttccagacattaaagatagtcatcttggggctggtcctgccgctgcttgtcatggtcatctgctactcgggaatcctaa aaactctgcttcggtgtcgaaatgagaagaagaggcacagggctgtgaggcttatcttcaccatcatgattgtttattttctcttctgggctc cctacaa SEQ ID NO: 8 Gaucher Disease: Strategy is to knock in GBA cDNA into Exon 3 of CCR5 safe harbor gene to overexpress GBA enzyme under macrophage-specific CD68 promoter. Left homology arro: 1-500 bp CD68 promoter: 507-969 bp GBA cDNA: 982-2589 bp BgH Poly A: 2616-2823 bp Right homology arm: 2841-3340 bp TTTCATGAATTCCCCCAACAGAGCCAAGCTCTCCATCTAGTGGACAGGGAAGCTA GCAGCAAACCTTCCCTTCACTACAAAACTTCATTGCTTGGCCAAAAAGAGAGTTA ATTCAATGTAGACATCTATGTAGGCAATTAAAAACCTATTGATGTATAAAACAGT TTGCATTCATGGAGGGCAACTAAATACATTCTAGGACTTTATAAAAGATCACTTT TTATTTATGCACAGGGTGGAACAAGATGGATTATCAAGTGTCAAGTCCAATCTAT GACATCAATTATTATACATCGGAGCCCTGCCAAAAAATCAATGTGAAGCAAATC GCAGCCCGCCTCCTGCCTCCGCTCTACTCACTGGTGTTCATCTTTGGTTTTGTGGG CAACATGCTGGTCATCCTCATCCTGATAAACTGCAAAAGGCTGAAGAGCATGACT GACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTTCCTTCTTACTGTCCC CTTCTctagaCTGTTCCCATAGCTACTTGCCACAACTGCCAAGCAAGTTTCGCTGAG TTTGACACATGGATCCCTGTGGATCAACTGCCCTAGGACTCCGTTTGCACCCATG TGACACTGTTGACTTTGCCCTGACGAAGCAGGGCCAACAGTCCCCTAACTTAATT ACAAAAACTAATGACTAAGAGAGAGGTGGCTAGAGCTGAGGCCCCTGAGTCAGG CTGTGGGTGGGATCATCTCCAGTACAGGAAGTGAGACTTTCATTTCCTCCTTTCC AAGAGAGGGCTGAGGGAGCAGGGTTGAGCAACTGGTGCAGACAGCCTAGCTGG ACTTTGGGTGAGGCGGTTCAGCCATATCGAATTCTGCTGGGGCTACTGGCAGGTA AGGAGGAAGGAGGCTGAGGGGAGGGGGCCCCTGGGAGGGAGCCTGCCCTGGGT TGCTAACCATCTCCTCTCTGCCAAAAGCCCAGGGGAttcgaaggatccatggagttttcaagtccttc cagagaggaatgtcccaagcctttgagtagggtaagcatcatggctggcagcctcacaggattgcttctacttcaggcagtgtcgtggg catcaggtgcccgcccctgcatccctaaaagcttcggctacagctcggtggtgtgtgtctgcaatgccacatactgtgactcctttgacc ccccgacctttcctgcccttggtaccttcagccgctatgagagtacacgcagtgggcgacggatggagctgagtatggggcccatcca ggctaatcacacgggcacaggcctgctactgaccctgcagccagaacagaagttccagaaagtgaagggatttggaggggccatga cagatgctgctgctctcaacatccttgccctgtcaccccctgcccaaaatttgctacttaaatcgtacttctctgaagaaggaatcggatat aacatcatccgggtacccatggccagctgtgacttctccatccgcacctacacctatgcagacacccctgatgatttccagttgcacaac ttcagcctcccagaggaagataccaagctcaagatacccctgattcaccgagccctgcagttggcccagcgtcccgtttcactccttgc cagcccctggacatcacccacttggctcaagaccaatggagcggtgaatgggaaggggtcactcaagggacagcccggagacatc taccaccagacctgggccagatactttgtgaagttcctggatgcctatgctgagcacaagttacagttctgggcagtgacagctgaaaat gagccttctgctgggctgttgagtggataccccttccagtgcctgggcttcacccctgaacatcagcgagacttcattgcccgtgaccta ggtcctaccctcgccaacagtactcaccacaatgtccgcctactcatgctggatgaccaacgcttgctgctgccccactgggcaaaggt ggtactgacagacccagaagcagctaaatatgttcatggcattgctgtacattggtacctggactttctggctccagccaaagccaccct aggggagacacaccgcctgttccccaacaccatgctctttgcctcagaggcctgtgtgggctccaagttctgggagcagagtgtgcg gctaggctcctgggatcgagggatgcagtacagccacagcatcatcacgaacctcctgtaccatgtggtcggctggaccgactggaa ccttgccctgaaccccgaaggaggacccaattgggtgcgtaactttgtcgacagtcccatcattgtagacatcaccaaggacacgtttta caaacagcccatgttctaccaccttggccacttcagcaagttcattcctgagggctcccagagagtggggctggttgccagtcagaag aacgacctggacgcagtggcactgatgcatcccgatggctctgctgttgtggtcgtgctaaaccgctcctctaaggatgtgcctcttacc atcaaggatcctgctgtgggcttcctggagacaalctcacctggctactccattcacacctacctgtggcgtcgccagTAGcTCG AGCCGCTGAtcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaagg tgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtgggg caggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctactagttgggctcactatgctgcc gcccagtgggactttggaaatacaatgtgtcaactcttgacagggctctattttataggcttcttctctggaatcttcttcatcatcctcctga caatcgataggtacctggctgtcgtccatgctgtgtttgctttaaaagccaggacggtcacctttggggtggtgacaagtgtgatcacttg ggtggtggctgtgtttgcgtctctcccaggaatcatctttaccagatctcaaaaagaaggtcttcattacacctgcagctctcattttccata cagtcagtatcaattctggaagaatttccagacattaaagatagtcatcttggggctggtcctgccgctgcttgtcatggtcatctgctact cgggaatcctaaaaactctgcttcggtgtcgaaatgagaagaagaggcacagggctgtgaggcttatcttcaccatcatgattgtttattt tctcttctgggctccctacaa

7. Exemplary Embodiments

Exemplary embodiments provided in accordance with the presently disclosed subject matter include, but are not limited to, the claims and the following embodiments:

-   -   1. A method of genetically modifying a cell from a subject with         a lysosomal storage disorder (LSD), the method comprising:     -   introducing into a cell isolated from the subject a single guide         RNA (sgRNA) targeting the CCR5 locus, an RNA-guided nuclease,         and a homologous donor template comprising a transgene encoding         a therapeutic protein that is absent or deficient in the         subject, wherein:     -   the sgRNA binds to the nuclease and directs it to a target         sequence at the CCR5 locus in the genome comprising the sequence         shown as SEQ ID NO:3 or SEQ ID NO:4, whereupon the nuclease         cleaves the CCR5 locus at the target sequence, wherein:     -   the homologous donor template comprises a first homology region         comprising the sequence of SEQ ID NO:1 or a fragment thereof to         one side of the transgene, and a second homology region         comprising the sequence of SEQ ID NO:2 or a fragment thereof to         the other side of the transgene, and the transgene is integrated         into the genome by homology directed recombination (HDR) at the         site of the cleaved CCR5 locus, and wherein     -   the integrated transgene directs the expression of the         therapeutic protein in the cell.     -   2. The method of embodiment 1, wherein the method further         comprises isolating the cell from the subject prior to the         introducing of the sgRNA, the RNA-guided nuclease, and the         homologous donor template.     -   3. The method of embodiment 1 or 2, wherein the sgRNA comprises         chemical modifications at one or more nucleotides.     -   4. The method of embodiment 3, wherein the sgRNA comprises         2′-O-methyl-3′-phosphorothioate (MS) modifications at one or         more nucleotides.     -   5. The method of embodiment 4, wherein the         2′-O-methyl-3′-phosphorothioate (MS) modifications are present         at the three terminal nucleotides of the 5′ and 3′ ends.     -   6. The method of any one of embodiments 1 to 5, wherein the         target sequence of the sgRNA comprises the sequence of SEQ ID         NO:3 or SEQ ID NO:4.     -   7. The method of embodiment 6, wherein the sgRNA comprises the         sequence of SEQ ID NO:5.     -   8. The method of any one of embodiments 1 to 7, wherein the         RNA-guided nuclease is Cas9.     -   9. The method of any one of embodiments 1 to 8, wherein the         sgRNA and the RNA-guided nuclease are introduced into the cell         as a ribonucleoprotein (RNP).     -   10. The method of embodiment 9, wherein the RNP is introduced         into the cell by electroporation.     -   11. The method of any one of embodiments 1 to 10, wherein the         transgene is present within an expression cassette.     -   12. The method of embodiment 11, wherein the expression cassette         comprises a coding sequence for the therapeutic protein,         operably linked to a promoter, and an exogenous polyadenylation         signal.     -   13. The method of embodiment 12, wherein the polyadenylation         signal is a bovine growth hormone polyadenylation signal.     -   14. The method of any one of embodiments 1 to 13, wherein the         homologous donor template is introduced into the cells using a         recombinant adeno-associated virus (rAAV) vector.     -   15. The method of embodiment 14, wherein the recombinant         adeno-associated virus is serotype 6 (rAAV6).     -   16. The method of any one of embodiments 1 to 15, wherein the         LSD is mucopolysaccharidosis type 1, and the therapeutic protein         is iduronidase.     -   17. The method of embodiment 16, wherein the transgene is part         of an expression cassette comprising the coding sequence for         iduronidase, operably linked to a phosphoglycerate kinase (PGK)         promoter or a spleen focus-forming virus (SFFV) promoter.     -   18. The method of embodiment 17, wherein the homologous donor         template comprises the sequence of SEQ ID NO: 6 or SEQ ID NO: 7.     -   19. The method of any of embodiments 16 to 18, wherein the cell         is a CD34⁺ hematopoietic stem and progenitor cell (HSPC).     -   20. The method of any one of embodiments 1 to 15, wherein the         LSD is Gaucher disease, and the therapeutic protein is         glucocerebrosidase.     -   21. The method of embodiment 20, wherein the transgene is part         of an expression cassette comprising the coding sequence for         glucocerebrosidase, operably linked to a CD68 promoter or         derivative thereof.     -   22. The method of embodiment 21, wherein the homologous donor         template comprises the sequence of SEQ ID NO: 8.     -   23. The method of any of embodiments 20 to 22, wherein the cell         is a CD34⁺ hematopoietic stem and progenitor cell (HSPC).     -   24. The method of any one of embodiments 1 to 15, wherein the         LSD is Krabbe disease, and the therapeutic protein is         galactocerebrosidase.     -   25. The method of embodiment 24, wherein the transgene is part         of an expression cassette comprising the coding sequence for         galactocerebrosidase, operably linked to a CD68 promoter or a         derivative thereof.     -   26. The method of embodiment 24 or 25, wherein the cell is a         CD34⁺ hematopoietic stem and progenitor cell (HSPC) or a         neuronal stem cell.     -   27. A method of treating a subject in need thereof with a         lysosomal storage disorder, comprising (i) genetically modifying         a cell from the subject using the method of any one of claims 1         to 26, and (ii) reintroducing the cell into the subject, wherein         the reintroducing is effective to treat the subject.     -   28. The method of embodiment 27, wherein the cell is         reintroduced into the subject by systemic transplantation.     -   29. The method of embodiment 27, wherein the cell is         reintroduced into the subject by local transplantation.     -   30. The method of embodiment 29, wherein the local         transplantation is intrafemoral or intrahepatic.     -   31. The method of any one of embodiments 27 to 30, wherein the         cell is cultured, selected, and/or induced to undergo         differentiation in vitro prior to being reintroduced into the         subject.     -   32. An sgRNA that specifically targets the CCR5 gene, wherein         the target sequence of the sgRNA comprises the nucleotide         sequence of SEQ ID NO:3 or SEQ ID NO:4.     -   33. The sgRNA of embodiment 32, wherein the sgRNA comprises the         nucleotide sequence of SEQ ID NO:5.     -   34. The sgRNA of embodiment 32 or 33, wherein the sgRNA         comprises chemical modifications at one or more nucleotides.     -   35. The sgRNA of embodiment 34, wherein the sgRNA comprises         2′-O-methyl-3′-phosphorothioate (MS) modifications at one or         more nucleotides.     -   36. The sgRNA of embodiment 35, wherein the         2′-O-methyl-3′-phosphorothioate (MS) modifications are present         at the three terminal nucleotides of the 5′ and 3′ ends.     -   37. A homologous donor template comprising:     -   (i) an expression cassette comprising: (a) a coding sequence for         a therapeutic protein, operably linked to (b) a promoter,         and (c) a polyadenylation signal at the 3′ end of the coding         sequence;     -   (ii) a first CCR5 homology region located to one side of the         expression cassette within the donor template, wherein the first         CCR5 homology region comprises SEQ ID NO:1 or a fragment         thereof; and     -   (iii) a second CCR5 homology region located to the other side of         the expression cassette within the donor template, wherein the         second CCR5 homology region comprises SEQ ID NO:2 or a fragment         thereof.     -   38. The donor template of embodiment 37, wherein the therapeutic         protein is iduronidase.     -   39. The donor template of embodiment 38, wherein the template         comprises the sequence shown as SEQ ID NO: 6 or SEQ ID NO: 7.     -   40. The donor template of embodiment 37, wherein the therapeutic         protein is glucocerebrosidase.     -   41. The donor template of embodiment 40, wherein the template         comprises the sequence shown as SEQ ID NO: 8.     -   42. The donor template of embodiment 37, wherein the therapeutic         protein is galactocerebrosidase.     -   43. An HSPC comprising the sgRNA of any one of embodiments 32 to         36, or a homologous donor template of any one of embodiments 37         to 42.     -   44. A genetically modified HSPC comprising an integrated         transgene at the CCR5 locus, wherein the integrated transgene         comprises a coding sequence for iduronidase, glucocerebrosidase,         or galactocerebrosidase.     -   45. The genetically modified HSPC of embodiment 44, wherein the         HSPC was modified using the method of any one of embodiments 1         to 26. 

What is claimed is:
 1. A method of genetically modifying a cell from a subject with a lysosomal storage disorder (LSD), the method comprising: introducing into a cell isolated from the subject a single guide RNA (sgRNA) targeting the CCR5 locus, an RNA-guided nuclease, and a homologous donor template comprising a transgene encoding a therapeutic protein that is absent or deficient in the subject, wherein: the sgRNA binds to the nuclease and directs it to a target sequence at the CCR5 locus in the genome comprising the sequence shown as SEQ ID NO:3 or SEQ ID NO:4, whereupon the nuclease cleaves the CCR5 locus at the target sequence, wherein: the homologous donor template comprises a first homology region comprising the sequence of SEQ ID NO:1 or a fragment thereof to one side of the transgene, and a second homology region comprising the sequence of SEQ ID NO:2 or a fragment thereof to the other side of the transgene, and the transgene is integrated into the genome by homology directed recombination (HDR) at the site of the cleaved CCR5 locus, and wherein the integrated transgene directs the expression of the therapeutic protein in the cell.
 2. The method of claim 1, wherein the method further comprises isolating the cell from the subject prior to the introducing of the sgRNA, the RNA-guided nuclease, and the homologous donor template.
 3. The method of claim 1, wherein the sgRNA comprises chemical modifications at one or more nucleotides.
 4. The method of claim 3, wherein the sgRNA comprises 2′-O-methyl-3′-phosphorothioate (MS) modifications at one or more nucleotides.
 5. The method of claim 4, wherein the 2′-O-methyl-3′-phosphorothioate (MS) modifications are present at the three terminal nucleotides of the 5′ and 3′ ends.
 6. The method of claim 1, wherein the target sequence of the sgRNA comprises the sequence of SEQ ID NO:3 or SEQ ID NO:4.
 7. The method of claim 6, wherein the sgRNA comprises the sequence of SEQ ID NO:5.
 8. The method of claim 1, wherein the RNA-guided nuclease is Cas9.
 9. The method of claim 1, wherein the sgRNA and the RNA-guided nuclease are introduced into the cell as a ribonucleoprotein (RNP).
 10. The method of claim 9, wherein the RNP is introduced into the cell by electroporation.
 11. The method of claim 1, wherein the transgene is present within an expression cassette.
 12. The method of claim 11, wherein the expression cassette comprises a coding sequence for the therapeutic protein, operably linked to a promoter, and an exogenous polyadenylation signal.
 13. The method of claim 12, wherein the polyadenylation signal is a bovine growth hormone polyadenylation signal.
 14. The method of claim 1, wherein the homologous donor template is introduced into the cells using a recombinant adeno-associated virus (rAAV) vector.
 15. The method of claim 14, wherein the recombinant adeno-associated virus is serotype 6 (rAAV6).
 16. The method of claim 1, wherein the LSD is mucopolysaccharidosis type 1, and the therapeutic protein is iduronidase.
 17. The method of claim 16, wherein the transgene is part of an expression cassette comprising the coding sequence for iduronidase, operably linked to a phosphoglycerate kinase (PGK) promoter or a spleen focus-forming virus (SFFV) promoter.
 18. The method of claim 17, wherein the homologous donor template comprises the sequence of SEQ ID NO: 6 or SEQ ID NO:
 7. 19. The method of claim 16, wherein the cell is a CD34⁺ hematopoietic stem and progenitor cell (HSPC).
 20. The method of claim 1, wherein the LSD is Gaucher disease, and the therapeutic protein is glucocerebrosidase.
 21. The method of claim 20, wherein the transgene is part of an expression cassette comprising the coding sequence for glucocerebrosidase, operably linked to a CD68 promoter or derivative thereof.
 22. The method of claim 21, wherein the homologous donor template comprises the sequence of SEQ ID NO:
 8. 23. The method of claim 20, wherein the cell is a CD34⁺ hematopoietic stem and progenitor cell (HSPC).
 24. The method of claim 1, wherein the LSD is Krabbe disease, and the therapeutic protein is galactocerebrosidase.
 25. The method of claim 24, wherein the transgene is part of an expression cassette comprising the coding sequence for galactocerebrosidase, operably linked to a CD68 promoter or a derivative thereof.
 26. The method of claim 24, wherein the cell is a CD34⁺ hematopoietic stem and progenitor cell (HSPC) or a neuronal stem cell.
 27. A method of treating a subject in need thereof with a lysosomal storage disorder, comprising (i) genetically modifying a cell from the subject using the method of any one of claims 1 to 26, and (ii) reintroducing the cell into the subject, wherein the reintroducing is effective to treat the subject.
 28. The method of claim 27, wherein the cell is reintroduced into the subject by systemic transplantation.
 29. The method of claim 27, wherein the cell is reintroduced into the subject by local transplantation.
 30. The method of claim 29, wherein the local transplantation is intrafemoral or intrahepatic.
 31. The method of claim 27, wherein the cell is cultured, selected, and/or induced to undergo differentiation in vitro prior to being reintroduced into the subject.
 32. An sgRNA that specifically targets the CCR5 gene, wherein the target sequence of the sgRNA comprises the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:4.
 33. The sgRNA of claim 32, wherein the sgRNA comprises the nucleotide sequence of SEQ ID NO:5.
 34. The sgRNA of claim 32, wherein the sgRNA comprises chemical modifications at one or more nucleotides.
 35. The sgRNA of claim 34, wherein the sgRNA comprises 2′-O-methyl-3′-phosphorothioate (MS) modifications at one or more nucleotides.
 36. The sgRNA of claim 35, wherein the 2′-O-methyl-3′-phosphorothioate (MS) modifications are present at the three terminal nucleotides of the 5′ and 3′ ends.
 37. A homologous donor template comprising: (i) an expression cassette comprising: (a) a coding sequence for a therapeutic protein, operably linked to (b) a promoter, and (c) a polyadenylation signal at the 3′ end of the coding sequence; (ii) a first CCR5 homology region located to one side of the expression cassette within the donor template, wherein the first CCR5 homology region comprises SEQ ID NO:1 or a fragment thereof; and (iii) a second CCR5 homology region located to the other side of the expression cassette within the donor template, wherein the second CCR5 homology region comprises SEQ ID NO:2 or a fragment thereof.
 38. The donor template of claim 37, wherein the therapeutic protein is iduronidase.
 39. The donor template of claim 38, wherein the template comprises the sequence shown as SEQ ID NO: 6 or SEQ ID NO:
 7. 40. The donor template of claim 37, wherein the therapeutic protein is glucocerebrosidase.
 41. The donor template of claim 40, wherein the template comprises the sequence shown as SEQ ID NO:
 8. 42. The donor template of claim 37, wherein the therapeutic protein is galactocerebrosidase.
 43. An HSPC comprising the sgRNA of claim 32 or a homologous donor template of claim
 37. 44. A genetically modified HSPC comprising an integrated transgene at the CCR5 locus, wherein the integrated transgene comprises a coding sequence for iduronidase, glucocerebrosidase, or galactocerebrosidase.
 45. The genetically modified HSPC of claim 44, wherein the HSPC was modified using the method of claim
 1. 